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Sample records for protein secretion system

  1. A light-triggered protein secretion system.

    PubMed

    Chen, Daniel; Gibson, Emily S; Kennedy, Matthew J

    2013-05-13

    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.

  2. 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

  3. A Brevibacillus choshinensis system that secretes cytoplasmic proteins.

    PubMed

    Horne, Irene; Williams, Michelle; Sutherland, Tara D; Russell, Robyn J; Oakeshott, John G

    2004-01-01

    Brevibacillus choshinensis has previously been shown to be a useful strain for the secretion of heterologous proteins via the Sec secretory pathway. This pathway involves the secretion of proteins prior to folding, whereas the alternative TAT (twin-arginine translocation) pathway enables pre-folded proteins to be secreted. We have modified the signal peptide of the Brevibacillus expression vector pNCMO2 to accommodate a Sec avoidance signal as well as the twin arginines required for secretion via the TAT system. Use of this modified signal peptide with the phosphotriesterase OpdA enabled B. choshinensis transformants to express and secrete the enzyme in an active and substantially pure form. The system was also used successfully to secrete two cytoplasmic proteins, the phosphotriesterase HocA from Pseudomonas monteilii and the phenylcarbamate-degrading enzyme, PCD, from Arthrobacter oxydans. The inhibitors carbonyl cyanide m-chlorophenyl hydrazine and sodium azide were used to confirm that secretion was occurring via the TAT secretion pathway. The modified B. choshinensis system we have developed may have general utility in secreting a wide range of heterologous proteins in active and conveniently processed form.

  4. Secretion of slow-folding proteins by a Type 1 secretion system.

    PubMed

    Schwarz, Christian K W; Lenders, Michael H H; Smits, Sander H J; Schmitt, Lutz

    2012-01-01

    Protein production through dedicated secretion systems might offer an potential alternative to the conventional cytoplasmical expression. The application of Type 1 secretion systems of Gram-negative bacteria, however, where often not successful in the past for a wide range of proteins. Recently, two studies using the E. coli maltose binding protein (MalE) and the rat intestinal fatty acid binding protein (IFABP) revealed a rational to circumvent these limitations. Here, wild-type passenger proteins were not secreted, while folding mutants with decreased folding kinetics were efficiently exported to the extracellular space. Subsequently, an one-step purification protocol yielded homogeneous and active protein. Taken together, theses two studies suggest that the introduction of slow-folding mutations into a protein sequence might be the key to use Type 1 secretion systems for the biotechnological production of proteins.

  5. A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins.

    PubMed

    Tejeda-Dominguez, Farid; Huerta-Cantillo, Jazmin; Chavez-Dueñas, Lucia; Navarro-Garcia, Fernando

    2017-03-28

    The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia, require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens.IMPORTANCE The type 3 secretion system is essential for injection of virulence factors, which are delivered directly into the cytosol of the host cells for usurping and subverting host processes. Recent studies have shown that these effectors proteins indeed travel inside an "injectisome" conduit through a single step of translocation by connecting the bacterium and host cell cytoplasms. However, all findings are not compatible with this model. For example, both YopH, a protein detected on the surface of Yersinia, and EspC, an autotransporter protein secreted by enteropathogenic E. coli, require a functional T3

  6. Characterization of Nops, nodulation outer proteins, secreted via the type III secretion system of NGR234.

    PubMed

    Marie, Corinne; Deakin, William J; Viprey, Virginie; Kopciñska, Joanna; Golinowski, Wladyslaw; Krishnan, Hari B; Perret, Xavier; Broughton, William J

    2003-09-01

    The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins). Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants. More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules. A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional. Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner. One of them was identified as NopA, a small 7-kDa protein. Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation. Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested. NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species. NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.

  7. A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins

    PubMed Central

    Tejeda-Dominguez, Farid; Huerta-Cantillo, Jazmin; Chavez-Dueñas, Lucia

    2017-01-01

    ABSTRACT The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia, require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens. PMID:28351918

  8. Identification of protein secretion systems in bacterial genomes.

    PubMed

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

    2016-03-16

    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 T6SS(iii) and T9SS were restricted to Bacteroidetes, and T6SS(ii) 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.

  9. Regulation of protein secretion by ... protein secretion?

    PubMed

    Atmakuri, Krishnamohan; Fortune, Sarah M

    2008-09-11

    Mycobacterium tuberculosis (Mtb) requires an alternative protein secretion system, ESX1, for virulence. Recently, Raghavan et al. (2008) reported a new regulatory circuit that may explain how ESX1 activity is controlled during infection. Mtb appears to regulate ESX1 by modulating transcription of associated genes rather than structural components of the secretion system itself.

  10. SecretEPDB: a comprehensive web-based resource for secreted effector proteins of the bacterial types III, IV and VI secretion systems

    PubMed Central

    An, Yi; Wang, Jiawei; Li, Chen; Revote, Jerico; Zhang, Yang; Naderer, Thomas; Hayashida, Morihiro; Akutsu, Tatsuya; Webb, Geoffrey I.; Lithgow, Trevor; Song, Jiangning

    2017-01-01

    Bacteria translocate effector molecules to host cells through highly evolved secretion systems. By definition, the function of these effector proteins is to manipulate host cell biology and the sequence, structural and functional annotations of these effector proteins will provide a better understanding of how bacterial secretion systems promote bacterial survival and virulence. Here we developed a knowledgebase, termed SecretEPDB (Bacterial Secreted Effector Protein DataBase), for effector proteins of type III secretion system (T3SS), type IV secretion system (T4SS) and type VI secretion system (T6SS). SecretEPDB provides enriched annotations of the aforementioned three classes of effector proteins by manually extracting and integrating structural and functional information from currently available databases and the literature. The database is conservative and strictly curated to ensure that every effector protein entry is supported by experimental evidence that demonstrates it is secreted by a T3SS, T4SS or T6SS. The annotations of effector proteins documented in SecretEPDB are provided in terms of protein characteristics, protein function, protein secondary structure, Pfam domains, metabolic pathway and evolutionary details. It is our hope that this integrated knowledgebase will serve as a useful resource for biological investigation and the generation of new hypotheses for research efforts aimed at bacterial secretion systems. PMID:28112271

  11. 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. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  12. 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

  13. 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

  14. 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

  15. Type III protein secretion systems in bacterial pathogens of animals and plants.

    PubMed

    Hueck, C J

    1998-06-01

    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

  16. SepD/SepL-dependent secretion signals of the type III secretion system translocator proteins in enteropathogenic Escherichia coli.

    PubMed

    Deng, Wanyin; Yu, Hong B; Li, Yuling; Finlay, B Brett

    2015-04-01

    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. 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 the host cells to

  17. New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens

    PubMed Central

    English, Grant; Trunk, Katharina; Rao, Vincenzo A; Srikannathasan, Velupillai; Hunter, William N; Coulthurst, Sarah J

    2012-01-01

    Protein secretion systems are critical to bacterial virulence and interactions with other organisms. The Type VI secretion system (T6SS) is found in many bacterial species and is used to target either eukaryotic cells or competitor bacteria. However, T6SS-secreted proteins have proven surprisingly elusive. Here, we identified two secreted substrates of the antibacterial T6SS from the opportunistic human pathogen, Serratia marcescens. Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS. Four related proteins encoded around the Ssp proteins (‘Rap’ proteins) included two specifically conferring self-resistance (‘immunity’) against T6SS-dependent Ssp1 or Ssp2 toxicity. Biochemical characterization revealed specific, tight binding between cognate Ssp–Rap pairs, forming complexes of 2:2 stoichiometry. The atomic structures of two Rap proteins were solved, revealing a novel helical fold, dependent on a structural disulphide bond, a structural feature consistent with their functional localization. Homologues of the Serratia Ssp and Rap proteins are found encoded together within other T6SS gene clusters, thus they represent founder members of new families of T6SS-secreted and cognate immunity proteins. We suggest that Ssp proteins are the original substrates of the S. marcescens T6SS, before horizontal acquisition of other T6SS-secreted toxins. Molecular insight has been provided into how pathogens utilize antibacterial T6SSs to overcome competitors and succeed in polymicrobial niches. PMID:22957938

  18. Secretome analysis uncovers an Hcp-family protein secreted via a type VI secretion system in Agrobacterium tumefaciens.

    PubMed

    Wu, Hung-Yi; Chung, Pei-Che; Shih, Hsiao-Wei; Wen, Sy-Ray; Lai, Erh-Min

    2008-04-01

    Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria.

  19. The Xanthomonas Hrp type III system secretes proteins from plant and mammalian bacterial pathogens

    PubMed Central

    Rossier, Ombeline; Wengelnik, Kai; Hahn, Karoline; Bonas, Ulla

    1999-01-01

    Studies of essential pathogenicity determinants in Gram-negative bacteria have revealed the conservation of type III protein secretion systems that allow delivery of virulence factors into host cells from plant and animal pathogens. Ten of 21 Hrp proteins of the plant pathogen Xanthomonas campestris pv. vesicatoria have been suggested to be part of a type III machinery. Here, we report the hrp-dependent secretion of two avirulence proteins, AvrBs3 and AvrRxv, by X. campestris pv. vesicatoria strains that constitutively express hrp genes. Secretion occurred without leakage of a cytoplasmic marker in minimal medium containing BSA, at pH 5.4. Secretion was strictly hrp-dependent because a mutant carrying a deletion in hrcV, a conserved hrp gene, did not secrete AvrBs3 and AvrRxv. Moreover, the Hrp system of X. campestris pv. vesicatoria was able to secrete proteins from two other plant pathogens: PopA, a protein secreted via the Hrp system in Ralstonia solanacearum, and AvrB, an avirulence protein from Pseudomonas syringae pv. glycinea. Interestingly, X. campestris pv. vesicatoria also secreted YopE, a type III-secreted cytotoxin of the mammalian pathogen Yersinia pseudotuberculosis in a hrp-dependent manner. YerA, a YopE-specific chaperone, was required for YopE stability but not for secretion in X. campestris pv. vesicatoria. Our results demonstrate the functional conservation of the type III system of X. campestris for secretion of proteins from both plant and mammalian pathogens and imply recognition of their respective secretion signals. PMID:10430949

  20. 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.

  1. 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

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

    SciTech Connect

    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.

  3. 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.

    2017-02-21

    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.

  4. Type II secretion system: a magic beanstalk or a protein escalator.

    PubMed

    Nivaskumar, Mangayarkarasi; Francetic, Olivera

    2014-08-01

    Type II protein secretion systems (T2SS) are molecular machines that promote specific transport of folded periplasmic proteins in Gram-negative bacteria, across a dedicated channel in the outer membrane. Secreted substrates, released to the milieu or displayed on the cell surface, contribute to bacterial adaptation to a range of habitats, from deep-sea waters to animal and plant tissues. The past decade has seen remarkable progress in structural, biochemical and functional analysis of T2SS and related systems, bringing new mechanistic insights into these dynamic complexes. This review focuses on recent advances in the field, and discusses open questions regarding the secretion mechanism. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. 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.

  6. 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

    2010-01-05

    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.

  7. 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

  8. Autotransporter protein secretion.

    PubMed

    Tame, Jeremy R H

    2011-12-01

    Autotransporter proteins are a large family of virulence factors secreted from Gram-negative bacteria by a unique mechanism. First described in the 1980s, these proteins have a C-terminal region that folds into a β-barrel in the bacterial outer membrane. The so-called passenger domain attached to this barrel projects away from the cell surface and may be liberated from the cell by self-cleavage or surface proteases. Although the majority of passenger domains have a similar β-helical structure, they carry a variety of sub-domains, allowing them to carry out widely differing functions related to pathogenesis. Considerable biochemical and structural characterisation of the barrel domain has shown that 'autotransporters' in fact require a conserved and essential protein complex in the outer membrane for correct folding. Although the globular domains of this complex projecting into the periplasmic space have also been structurally characterised, the overall secretion pathway of the autotransporters remains highly puzzling. It was presumed for many years that the passenger domain passed through the centre of the barrel domain to reach the cell surface, driven at least in part by folding. This picture is complicated by conflicting data, and there is currently little hard information on the true nature of the secretion intermediates. As well as their medical importance therefore, autotransporters are proving to be an excellent system to study the folding and membrane insertion of outer membrane proteins in general. This review focuses on structural aspects of autotransporters; their many functions in pathogenesis are beyond its scope.

  9. Identification of Protein Secretion Systems in Bacterial Genomes Using MacSyFinder.

    PubMed

    Abby, Sophie S; Rocha, Eduardo P C

    2017-01-01

    Protein secretion systems are complex molecular machineries that translocate proteins through the outer membrane, and sometimes through multiple other barriers. They have evolved by co-option of components from other envelope-associated cellular machineries, making them sometimes difficult to identify and discriminate. Here, we describe how to identify protein secretion systems in bacterial genomes using MacSyFinder. This flexible computational tool uses the knowledge stemming from experimental studies to identify homologous systems in genome data. It can be used with a set of predefined models-"TXSScan"-to identify all major secretion systems of diderm bacteria (i.e., with inner and with LPS-containing outer membranes). For this, it identifies and clusters colocalized components of secretion systems using sequence similarity searches with hidden Markov model protein profiles. Finally, it checks whether the genetic content and organization of clusters satisfy the constraints of the model. TXSScan models can be customized to search for variants of known systems. The models can also be built from scratch to identify novel systems. In this chapter, we describe a complete pipeline of analysis, including the identification of a reference set of experimentally studied systems, the identification of components and the construction of their protein profiles, the definition of the models, their optimization, and, finally, their use as tools to search genomic data.

  10. A Reporter System to Study Unconventional Secretion of Proteins Avoiding N-Glycosylation in Ustilago maydis.

    PubMed

    Stock, Janpeter; Terfrüchte, Marius; Schipper, Kerstin

    2016-01-01

    Unconventional secretion of proteins in eukaryotes is characterized by the circumvention of the Endoplasmic Reticulum (ER). As a consequence proteins exported by unconventional pathways lack N-glycosylation, a post-transcriptional modification that is initiated in the ER during classical secretion. We are exploiting the well-established enzyme β-glucuronidase (GUS) to assay unconventional protein secretion (UPS). This bacterial protein is perfectly suited for this purpose because it carries a eukaryotic N-glycosylation motif. Modification of this residue by attachment of sugar moieties during the passage of the ER apparently causes a very strong reduction in GUS activity. Hence, this enzyme can only be secreted in an active state, if the export mechanism does not involve ER passage. Here, we describe a reporter system applied in the corn smut fungus Ustilago maydis that is based on this observation and can be used to test if candidate proteins are secreted to the culture supernatant via alternative pathways avoiding N-glycosylation. Importantly, this system is the basis for the establishment of genetic screens providing mechanistic insights into unknown UPS pathways in the future.

  11. 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.

  12. Campylobacter fetus Surface Layer Proteins Are Transported by a Type I Secretion System

    PubMed Central

    Thompson, Stuart A.; Shedd, Omer L.; Ray, Kevin C.; Beins, Michael H.; Jorgensen, Jesse P.; Blaser, Martin J.

    1998-01-01

    The virulence of Campylobacter fetus, a bacterial pathogen of ungulates and humans, is mediated in part by the presence of a paracrystalline surface layer (S-layer) that confers serum resistance. The subunits of the S-layer are S-layer proteins (SLPs) that are secreted in the absence of an N-terminal signal sequence and attach to either type A or B C. fetus lipopolysaccharide in a serospecific manner. Antigenic variation of multiple SLPs (encoded by sapA homologs) of type A strain 23D occurs by inversion of a promoter-containing DNA element flanked by two sapA homologs. Cloning and sequencing of the entire 6.2-kb invertible region from C. fetus 23D revealed a probable 5.6-kb operon of four overlapping genes (sapCDEF, with sizes of 1,035, 1,752, 1,284, and 1,302 bp, respectively) transcribed in the opposite direction from sapA. The four genes also were present in the invertible region of type B strain 84-107 and were virtually identical to their counterparts in the type A strain. Although SapC had no database homologies, SapD, SapE, and SapF had predicted amino acid homologies with type I protein secretion systems (typified by Escherichia coli HlyBD/TolC or Erwinia chrysanthemi PrtDEF) that utilize C-terminal secretion signals to mediate the secretion of hemolysins, leukotoxins, or proteases from other bacterial species. Analysis of the C termini of four C. fetus SLPs revealed conserved structures that are potential secretion signals. A C. fetus sapD mutant neither produced nor secreted SLPs. E. coli expressing C. fetus sapA and sapCDEF secreted SapA, indicating that the sapCDEF genes are sufficient for SLP secretion. C. fetus SLPs therefore are transported to the cell surface by a type I secretion system. PMID:9851986

  13. Galectin-3 directs antimicrobial guanylate binding proteins to vacuoles furnished with bacterial secretion systems

    PubMed Central

    Feeley, Eric M.; Pilla-Moffett, Danielle M.; Zwack, Erin E.; Piro, Anthony S.; Finethy, Ryan; Kolb, Joseph P.; Martinez, Jennifer; Brodsky, Igor E.; Coers, Jörn

    2017-01-01

    Many invasive bacteria establish pathogen-containing vacuoles (PVs) as intracellular niches for microbial growth. Immunity to these infections is dependent on the ability of host cells to recognize PVs as targets for host defense. The delivery of several host defense proteins to PVs is controlled by IFN-inducible guanylate binding proteins (GBPs), which themselves dock to PVs through poorly characterized mechanisms. Here, we demonstrate that GBPs detect the presence of bacterial protein secretion systems as “patterns of pathogenesis” associated with PVs. We report that the delivery of GBP2 to Legionella-containing vacuoles is dependent on the bacterial Dot/Icm secretion system, whereas the delivery of GBP2 to Yersinia-containing vacuoles (YCVs) requires hypersecretion of Yersinia translocon proteins. We show that the presence of bacterial secretion systems directs cytosolic carbohydrate-binding protein Galectin-3 to PVs and that the delivery of GBP1 and GBP2 to Legionella-containing vacuoles or YCVs is substantially diminished in Galectin-3–deficient cells. Our results illustrate that insertion of bacterial secretion systems into PV membranes stimulates Galectin-3–dependent recruitment of antimicrobial GBPs to PVs as part of a coordinated host defense program. PMID:28193861

  14. Galectin-3 directs antimicrobial guanylate binding proteins to vacuoles furnished with bacterial secretion systems.

    PubMed

    Feeley, Eric M; Pilla-Moffett, Danielle M; Zwack, Erin E; Piro, Anthony S; Finethy, Ryan; Kolb, Joseph P; Martinez, Jennifer; Brodsky, Igor E; Coers, Jörn

    2017-02-28

    Many invasive bacteria establish pathogen-containing vacuoles (PVs) as intracellular niches for microbial growth. Immunity to these infections is dependent on the ability of host cells to recognize PVs as targets for host defense. The delivery of several host defense proteins to PVs is controlled by IFN-inducible guanylate binding proteins (GBPs), which themselves dock to PVs through poorly characterized mechanisms. Here, we demonstrate that GBPs detect the presence of bacterial protein secretion systems as "patterns of pathogenesis" associated with PVs. We report that the delivery of GBP2 to Legionella-containing vacuoles is dependent on the bacterial Dot/Icm secretion system, whereas the delivery of GBP2 to Yersinia-containing vacuoles (YCVs) requires hypersecretion of Yersinia translocon proteins. We show that the presence of bacterial secretion systems directs cytosolic carbohydrate-binding protein Galectin-3 to PVs and that the delivery of GBP1 and GBP2 to Legionella-containing vacuoles or YCVs is substantially diminished in Galectin-3-deficient cells. Our results illustrate that insertion of bacterial secretion systems into PV membranes stimulates Galectin-3-dependent recruitment of antimicrobial GBPs to PVs as part of a coordinated host defense program.

  15. Use of Transcriptional Control to Increase Secretion of Heterologous Proteins in T3S Systems.

    PubMed

    Metcalf, Kevin J; Tullman-Ercek, Danielle

    2017-01-01

    Heterologous proteins can be produced in a bacterial host and purified from the cellular constituents. Secretion of the protein of interest to the extracellular space simplifies the purification process and is thought to alleviate toxicity problems associated with intracellular accumulation of the protein of interest. In this protocol, we describe a strategy to engineer protein secretion in a bacterial culture using transcriptional control. The transcription factor HilA is inducibly produced to control production of the secretion machine, and in turn signals the production and secretion of a protein of interest. This allows for high titer of secreted protein in optimized culturing conditions and the effect is observed with all proteins tested.

  16. Secreted protein-based reporter systems for monitoring inflammatory events: critical interference by endoplasmic reticulum stress.

    PubMed

    Hiramatsu, Nobuhiko; Kasai, Ayumi; Hayakawa, Kunihiro; Nagai, Kaoru; Kubota, Takeo; Yao, Jian; Kitamura, Masanori

    2006-08-31

    A number of recent reports have used secreted protein-based reporter assays for monitoring intercellular and intracellular events involved in inflammation. However, we found that these assay systems are critically affected by endoplasmic reticulum (ER) stress. When reporter mesangial cells that express secreted alkaline phosphatase (SEAP) under the control of NF-kappaB were exposed to IL-1beta or TNF-alpha, induction of SEAP activity was markedly reduced under ER stress conditions. Downregulation of SEAP activity was observed regardless of cell types and type of regulatory elements; e.g., when reporter hepatocytes that express SEAP under the control of the dioxin responsive elements were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin or benzo[a]pyrene, ER stress similarly suppressed the increase in SEAP activity despite its transcriptional upregulation. Activity of constitutively expressed SEAP in various cells was also reduced by ER stress in a magnitude-dependent manner, and it was associated with disturbed subcellular transport of SEAP to the Golgi. Furthermore, interference by ER stress was similarly observed in other reporter assay using secreted luciferase. These results evidenced critical interference by ER stress in secreted protein-based reporter systems. The suppression of reporter responses by ER stress should be considered carefully for experimental design and interpretation of data when secreted protein-based reporter systems are used for investigation.

  17. 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

  18. Identification of genes encoding the type IX secretion system and secreted proteins in Flavobacterium columnare IA-S-4

    USDA-ARS?s Scientific Manuscript database

    Flavobacterium columnare, a member of the phylum Bacteroidetes, causes columnaris disease in wild and aquaculture-reared freshwater fish. The mechanisms responsible for columnaris disease are not known. Many members of the phylum Bacteroidetes use type IX secretion systems (T9SSs) to secrete enzymes...

  19. Analysis of secreted proteins.

    PubMed

    Severino, Valeria; Farina, Annarita; Chambery, Angela

    2013-01-01

    Most biological processes including growth, proliferation, differentiation, and apoptosis are coordinated by tightly regulated signaling pathways, which also involve secreted proteins acting in an autocrine and/or paracrine manner. In addition, extracellular signaling molecules affect local niche biology and influence the cross-talking with the surrounding tissues. The understanding of this molecular language may provide an integrated and broader view of cellular regulatory networks under physiological and pathological conditions. In this context, the profiling at a global level of cell secretomes (i.e., the subpopulations of a proteome secreted from the cell) has become an active area of research. The current interest in secretome research also deals with its high potential for the biomarker discovery and the identification of new targets for therapeutic strategies. Several proteomic and mass spectrometry platforms and methodologies have been applied to secretome profiling of conditioned media of cultured cell lines and primary cells. Nevertheless, the analysis of secreted proteins is still a very challenging task, because of the technical difficulties that may hamper the subsequent mass spectrometry analysis. This chapter describes a typical workflow for the analysis of proteins secreted by cultured cells. Crucial issues related to cell culture conditions for the collection of conditioned media, secretome preparation, and mass spectrometry analysis are discussed. Furthermore, an overview of quantitative LC-MS-based approaches, computational tools for data analysis, and strategies for validation of potential secretome biomarkers is also presented.

  20. Flavobacterium johnsoniae PorV is required for secretion of a subset of proteins targeted to the type IX secretion system.

    PubMed

    Kharade, Sampada S; McBride, Mark J

    2015-01-01

    Flavobacterium johnsoniae exhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion, and F. johnsoniae has orthologs of these. Mutations in F. johnsoniae porU and porV were constructed to determine if they function in secretion. Cells of a porV deletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and the porV mutant exhibited gliding motility and formed spreading colonies. However, the porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.

  1. 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

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

    PubMed

    Tomek, M B; Neumann, L; Nimeth, I; Koerdt, A; Andesner, P; Messner, P; Mach, L; Potempa, J S; Schäffer, C

    2014-12-01

    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 two-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 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 being 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 fewer voids compared with 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 by the two S-layer proteins. In addition, T9SS protein translocation is decoupled from O-glycan attachment in T. forsythia.

  3. The type III protein secretion system contributes to Xanthomonas citri subsp. citri biofilm formation

    PubMed Central

    2014-01-01

    Background Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system is involved in multicellular processes, such as bacterial biofilm formation has not been elucidated. Here, the phytopathogen Xanthomonas citri subsp. citri (X. citri) was used as a model to gain further insights about the role of the T3SS in biofilm formation. Results The capacity of biofilm formation of different X. citri T3SS mutants was compared to the wild type strain and it was observed that this secretion system was necessary for this process. Moreover, the T3SS mutants adhered proficiently to leaf surfaces but were impaired in leaf-associated growth. A proteomic study of biofilm cells showed that the lack of the T3SS causes changes in the expression of proteins involved in metabolic processes, energy generation, exopolysaccharide (EPS) production and bacterial motility as well as outer membrane proteins. Furthermore, EPS production and bacterial motility were also altered in the T3SS mutants. Conclusions Our results indicate a novel role for T3SS in X. citri in the modulation of biofilm formation. Since this process increases X. citri virulence, this study reveals new functions of T3SS in pathogenesis. PMID:24742141

  4. Chlamydia trachomatis secretion of hypothetical protein CT622 into host cell cytoplasm via a secretion pathway that can be inhibited by the type III secretion system inhibitor compound 1

    PubMed Central

    Gong, Siqi; Lei, Lei; Chang, Xiaotong; Belland, Robert; Zhong, Guangming

    2011-01-01

    Using antibodies raised with C. trachomatis fusion proteins, we localized a hypothetical protein encoded by the ORF ct622 in the cytoplasm of C. trachomatis-infected mammalian cells. The detection was specific since the antibody labelling of CT622 protein was removed by preabsorption with CT622 but not other fusion proteins. We similarly confirmed that CT621, a known secretion protein encoded by a hypothetical ORF downstream of ct622, was secreted into host cell cytosol. Proteins CT622 and CT621 displayed a similar secretion pattern, with both intra-inclusion and host cell cytosol localization, that was distinct from that of CPAF (chlamydial protease/proteasome-like activity factor). However, the expression and secretion kinetics differed significantly between CT622 and CT621: CT622 mRNA was detected at 2 h, protein at 6 h and secretion of protein into host cell cytoplasm at 36 h post-infection, while CT621 mRNA was detected at 8 h, protein at 16 h and secretion at 24 h. The secretion of both CT622 and CT621 was blocked by N′-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide (compound 1), an inhibitor known to target the type III secretion system of bacteria. These results suggest that CT621 and CT622 may fulfil different functions during chlamydial intracellular growth. Further characterization of these proteins may generate important information for understanding chlamydial pathogenesis. PMID:21233161

  5. 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

    2015-10-12

    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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Secretion by numbers: protein traffic in prokaryotes

    PubMed Central

    Economou, Anastasias; Christie, Peter J.; Fernandez, Rachel C.; Palmer, Tracy; Plano, Greg V.; Pugsley, Anthony P.

    2013-01-01

    Summary Almost all aspects of protein traffic in bacteria were covered at the ASM-FEMS meeting on the topic in Iraklio, Crete in May 2006. The studies presented ranged from mechanistic analysis of specific events leading proteins to their final destinations to the physiological roles of the targeted proteins. Among the highlights from the meeting that are reviewed here are the molecular dynamics of SecA protein, membrane protein insertion, type III secretion needles and chaperones, type IV secretion, the two partner and autosecretion systems, the ‘secretion competent state’, and the recently discovered type VI secretion system. PMID:17020575

  7. 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

  8. 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.

  9. The Salmonella Type III Secretion System Inner Rod Protein PrgJ Is Partially Folded*

    PubMed Central

    Zhong, Dalian; Lefebre, Matthew; Kaur, Kawaljit; McDowell, Melanie A.; Gdowski, Courtney; Jo, Sunhwan; Wang, Yu; Benedict, Stephen H.; Lea, Susan M.; Galan, Jorge E.; De Guzman, Roberto N.

    2012-01-01

    The type III secretion system (T3SS) is essential in the pathogenesis of many bacteria. The inner rod is important in the assembly of the T3SS needle complex. However, the atomic structure of the inner rod protein is currently unknown. Based on computational methods, others have suggested that the Salmonella inner rod protein PrgJ is highly helical, forming a folded 3 helix structure. Here we show by CD and NMR spectroscopy that the monomeric form of PrgJ lacks a tertiary structure, and the only well-structured part of PrgJ is a short α-helix at the C-terminal region from residues 65–82. Disruption of this helix by glycine or proline mutation resulted in defective assembly of the needle complex, rendering bacteria incapable of secreting effector proteins. Likewise, CD and NMR data for the Shigella inner rod protein MxiI indicate this protein lacks a tertiary structure as well. Our results reveal that the monomeric forms of the T3SS inner rod proteins are partially folded. PMID:22654099

  10. Secretion of the Rhizobium leguminosarum nodulation protein NodO by haemolysin-type systems.

    PubMed

    Scheu, A K; Economou, A; Hong, G F; Ghelani, S; Johnston, A W; Downie, J A

    1992-01-01

    The Rhizobium leguminosarum biovar viciae nodulation protein NodO is partially homologous to haemolysin of Escherichia coli and, like haemolysin, is secreted into the growth medium. The NodO protein can be secreted by a strain of E. coli carrying the cloned nodO gene plus the haemolysin secretion genes hlyBD, in a process that also requires the outer membrane protein encoded by tolC. The related protease secretion genes, prtDEF, from Erwinia chrysanthemi also enable E. coli to secrete NodO. The Rhizobium genes encoding the proteins required for NodO secretion are unlinked to nodO and are unlike other nod genes, since they do not require flavonoids or NodO for their expression. Although proteins similar to NodO were not found in rhizobia other than R. leguminosarum bv. viciae, several rhizobia and an Agrobacterium strain containing the cloned nodO gene were found to have the ability to secrete NodO. These observations indicate that a wide range of the Rhizobiaceae have a protein secretion mechanism analogous to that which secretes haemolysin and related toxins and proteases in the ENterobacteriaceae.

  11. Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae.

    PubMed

    Unterweger, Daniel; Kostiuk, Benjamin; Ötjengerdes, Rina; Wilton, Ashley; Diaz-Satizabal, Laura; Pukatzki, Stefan

    2015-08-13

    Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines. © 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  12. 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

  13. Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in the Yersinia Type III Secretion System.

    PubMed

    Ho, Oanh; Rogne, Per; Edgren, Tomas; Wolf-Watz, Hans; Login, Frédéric H; Wolf-Watz, Magnus

    2017-02-24

    Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia, the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscUC binds to the inner rod protein YscI with a dissociation constant (Kd ) of 3.8 μm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria.

  14. Acylation of the Type 3 Secretion System Translocon Using a Dedicated Acyl Carrier Protein

    PubMed Central

    Agrebi, Rym; Canestrari, Mickaël J.; Mignot, Tâm; Lebrun, Régine; Bouveret, Emmanuelle

    2017-01-01

    Bacterial pathogens often deliver effectors into host cells using type 3 secretion systems (T3SS), the extremity of which forms a translocon that perforates the host plasma membrane. The T3SS encoded by Salmonella pathogenicity island 1 (SPI-1) is genetically associated with an acyl carrier protein, IacP, whose role has remained enigmatic. In this study, using tandem affinity purification, we identify a direct protein-protein interaction between IacP and the translocon protein SipB. We show, by mass spectrometry and radiolabelling, that SipB is acylated, which provides evidence for a modification of the translocon that has not been described before. A unique and conserved cysteine residue of SipB is identified as crucial for this modification. Although acylation of SipB was not essential to virulence, we show that this posttranslational modification promoted SipB insertion into host-cell membranes and pore-forming activity linked to the SPI-1 T3SS. Cooccurrence of acyl carrier and translocon proteins in several γ- and β-proteobacteria suggests that acylation of the translocon is conserved in these other pathogenic bacteria. These results also indicate that acyl carrier proteins, known for their involvement in metabolic pathways, have also evolved as cofactors of new bacterial protein lipidation pathways. PMID:28085879

  15. 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.

  16. A Vector System for ABC Transporter-Mediated Secretion and Purification of Recombinant Proteins in Pseudomonas Species

    PubMed Central

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

    2014-01-01

    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

  17. Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium.

    PubMed

    Michel, Gérard P F; Aguzzi, Anthony; Ball, Geneviève; Soscia, Chantal; Bleves, Sophie; Voulhoux, Romé

    2011-07-01

    Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

  18. Identification of Novel Protein-Protein Interactions of Yersinia pestis Type III Secretion System by Yeast Two Hybrid System

    PubMed Central

    Tang, Liujun; Wang, Jian; Ke, Yuehua; Guo, Zhaobiao; Yang, Xiaoming; Yang, Ruifu; Du, Zongmin

    2013-01-01

    Type III secretion system (T3SS) of the plague bacterium Y. pestis encodes a syringe-like structure consisting of more than 20 proteins, which can inject virulence effectors into host cells to modulate the cellular functions. Here in this report, interactions among the possible components in T3SS of Yersinia pestis were identified using yeast mating technique. A total of 57 genes, including all the pCD1-encoded genes except those involved in plasmid replication and partition, pseudogenes, and the putative transposase genes, were subjected to yeast mating analysis. 21 pairs of interaction proteins were identified, among which 9 pairs had been previously reported and 12 novel pairs were identified in this study. Six of them were tested by GST pull down assay, and interaction pairs of YscG-SycD, YscG-TyeA, YscI-YscF, and YopN-YpCD1.09c were successfully validated, suggesting that these interactions might play potential roles in function of Yersinia T3SS. Several potential new interactions among T3SS components could help to understand the assembly and regulation of Yersinia T3SS. PMID:23349800

  19. Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD*

    PubMed Central

    Chatterjee, Abhishek; Caballero-Franco, Celia; Bakker, Dannika; Totten, Stephanie; Jardim, Armando

    2015-01-01

    Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits. PMID:26324713

  20. 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. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  1. Cells from the skin of patients with systemic sclerosis secrete chitinase 3-like protein 1

    PubMed Central

    Ho, Yuen Yee; Baron, Murray; Recklies, Anneliese D.; Roughley, Peter J.; Mort, John S.

    2014-01-01

    Background The chitinase-like protein, Chi3L1, is associated with increased fibrotic activity as well as inflammatory processes. The capacity of skin cells from systemic sclerosis (SSc) patients to produce Chi3L1, and the stimulation of its synthesis by cytokines or growth factors known to be associated with SSc, was investigated. Methods Cells were isolated from forearm and/or abdomen skin biopsies taken from SSc patients and normal individuals and stimulated with cytokines and growth factors to assess Chi3L1 expression. Chi3L1-expressing cells were characterized by immunohistochemical staining. Results Chi3L1 was not secreted by skin cells from normal individuals nor was its synthesis induced by any of the cytokines or growth factors investigated. In contrast, Chi3L1 secretion was induced by OSM or IL-1 in cells from all forearm biopsies of SSc patients, and endogenous secretion in the absence of cytokines was detected in several specimens. Patients with Chi3L1-producing cells at both the arm and abdomen had a disease duration of less than 3 years. Endogenous Chi3L1 production was not a property of the major fibroblast population nor of myofibroblasts, but rather was related to the presence of stem-like cells not present in normal skin. Other cells, however, contributed to the upregulation of Chi3L1 by OSM. Conclusions The emergence of cells primed to respond to OSM with increased Chi3L1 production appears to be associated with pathological processes active in SSc. General significance The presence of progenitor cells expressing the chilectin Chi3L1 in SSc skin appears to play a role in the initiation of the disease process. PMID:26675476

  2. PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System

    PubMed Central

    Veith, Paul D.; Butler, Catherine A.; Nor Muhammad, Nor A.; Chen, Yu-Yen; Slakeski, Nada; Peng, Benjamin; Zhang, Lianyi; Dashper, Stuart G.; Cross, Keith J.; Cleal, Steven M.; Moore, Caroline; Reynolds, Eric C.

    2016-01-01

    Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS. PMID:27711252

  3. A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins

    PubMed Central

    Kakitani, Makoto; Oshima, Takeshi; Horikoshi, Kaori; Yoshitome, Tetsuo; Ueda, Akiko; Kajikawa, Miwa; Iba, Yumi; Ozone, Yoshinao; Ijima, Yuki; Yoshino, Tohko; Itoh, Mikiko; Seki, Sachiko; Aoki, Ayako; Ishihara, Toshie; Shionoya, Michiyo; Makino, Utako; Kitada, Rina; Ohguma, Atsuko; Ohta, Takami; Yoshida, Yoshimasa; Kudoh, Hiroe; Hanaoka, Kazunori; Sibuya, Kazunori; Ishida, Isao; Kakeda, Minoru; Yagi, Mikio; Yoneya, Takashi; Tomizuka, Kazuma

    2005-01-01

    A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) κ locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igκ region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras. PMID:15914664

  4. 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

  5. The type 3 protein secretion system of Cupriavidus taiwanensis strain LMG19424 compromises symbiosis with Leucaena leucocephala.

    PubMed

    Saad, Maged M; Crèvecoeur, Michèle; Masson-Boivin, Catherine; Perret, Xavier

    2012-10-01

    Cupriavidus taiwanensis forms proficient symbioses with a few Mimosa species. Inactivation of a type III protein secretion system (T3SS) had no effect on Mimosa pudica but allowed C. taiwanensis to establish chronic infections and fix nitrogen in Leucaena leucocephala. Unlike what was observed for other rhizobia, glutamate rather than plant flavonoids mediated transcriptional activation of this atypical T3SS.

  6. The Type 3 Protein Secretion System of Cupriavidus taiwanensis Strain LMG19424 Compromises Symbiosis with Leucaena leucocephala

    PubMed Central

    Saad, Maged M.; Crèvecoeur, Michèle; Masson-Boivin, Catherine

    2012-01-01

    Cupriavidus taiwanensis forms proficient symbioses with a few Mimosa species. Inactivation of a type III protein secretion system (T3SS) had no effect on Mimosa pudica but allowed C. taiwanensis to establish chronic infections and fix nitrogen in Leucaena leucocephala. Unlike what was observed for other rhizobia, glutamate rather than plant flavonoids mediated transcriptional activation of this atypical T3SS. PMID:22865066

  7. 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

  8. Type VI secretion system.

    PubMed

    Salomon, Dor; Orth, Kim

    2015-03-30

    Bacteria employ a variety of tools to survive in a competitive environment. Salomon and Orth describe one such tool-the Type 6 Secretion Systems used by bacteria to deliver a variety of toxins into competing cells.

  9. The Pseudomonas aeruginosa patatin-like protein PlpD is the archetype of a novel Type V secretion system.

    PubMed

    Salacha, Richard; Kovacić, Filip; Brochier-Armanet, Céline; Wilhelm, Susanne; Tommassen, Jan; Filloux, Alain; Voulhoux, Romé; Bleves, Sophie

    2010-06-01

    We discovered a novel secreted protein by Pseudomonas aeruginosa, PlpD, as a member of the bacterial lipolytic enzyme family of patatin-like proteins (PLPs). PlpD is synthesized as a single molecule consisting of a secreted domain fused to a transporter domain. The N-terminus of PlpD includes a classical signal peptide followed by the four PLP conserved blocks that account for its lipase activity. The C-terminus consists of a POTRA (polypeptide transport-associated) motif preceding a putative 16-stranded beta-barrel similar to those of TpsB transporters of Type Vb secretion system. We showed that the C-terminus remains inserted into the outer membrane while the patatin moiety is secreted. The association between a TpsB component and a passenger protein is a unique hybrid organization that we propose to classify as Type Vd. More than 200 PlpD orthologues exist among pathogenic and environmental bacteria, which suggests that bacteria secrete numerous PLPs using this newly defined mechanism.

  10. A homologous production system for Trichoderma reesei secreted proteins in a cellulase-free background.

    PubMed

    Uzbas, Fatma; Sezerman, Ugur; Hartl, Lukas; Kubicek, Christian P; Seiboth, Bernhard

    2012-02-01

    Recent demands for the production of biofuels from lignocellulose led to an increased interest in engineered cellulases from Trichoderma reesei or other fungal sources. While the methods to generate such mutant cellulases on DNA level are straightforward, there is often a bottleneck in their production since a correct posttranslational processing of these enzymes is needed to obtain highly active enzymes. Their production and subsequent enzymatic analysis in the homologous host T. reesei is, however, often disturbed by the concomitant production of other endogenous cellulases. As a useful alternative, we tested the production of cellulases in T. reesei in a genetic background where cellulase formation has been impaired by deletion of the major cellulase transcriptional activator gene xyr1. Three cellulase genes (cel7a, cel7b, and cel12a) were expressed under the promoter regions of the two highly expressed genes tef1 (encoding translation elongation factor 1-alpha) or cdna1 (encoding the hypothetical protein Trire2:110879). When cultivated on D: -glucose as carbon source, the Δxyr1 strain secreted all three cellulases into the medium. Related to the introduced gene copy number, the cdna1 promoter appeared to be superior to the tef1 promoter. No signs of proteolysis were detected, and the individual cellulases could be assayed over a background essentially free of other cellulases. Hence this system can be used as a vehicle for rapid and high-throughput testing of cellulase muteins in a homologous background.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  12. New protein-protein interactions identified for the regulatory and structural components and substrates of the type III Secretion system of the phytopathogen Xanthomonas axonopodis Pathovar citri.

    PubMed

    Alegria, Marcos C; Docena, Cassia; Khater, Leticia; Ramos, Carlos H I; da Silva, Ana C R; Farah, Chuck S

    2004-09-01

    We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv. citri. Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators, and substrates of the type III secretion system coded by the hrp (for hypersensitive response and pathogenicity), hrc (for hrp conserved), and hpa (for hrp associated) genes. We have identified several previously uncharacterized interactions involving (i) HrpG, a two-component system response regulator responsible for the expression of X. axonopodis pv. citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; (ii) HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV; (iii) HrpB1, HrpD6, and HrpW; and (iv) HrpB2 and HrcU. Homotropic interactions were also identified for the ATPase HrcN. These newly identified protein-protein interactions increase our understanding of the functional integration of phytopathogen-specific type III secretion system components and suggest new hypotheses regarding the molecular mechanisms underlying Xanthomonas pathogenicity.

  13. New Protein-Protein Interactions Identified for the Regulatory and Structural Components and Substrates of the Type III Secretion System of the Phytopathogen Xanthomonas axonopodis Pathovar citri

    PubMed Central

    Alegria, Marcos C.; Docena, Cassia; Khater, Leticia; Ramos, Carlos H. I.; da Silva, Ana C. R.; Farah, Chuck S.

    2004-01-01

    We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv. citri. Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators, and substrates of the type III secretion system coded by the hrp (for hypersensitive response and pathogenicity), hrc (for hrp conserved), and hpa (for hrp associated) genes. We have identified several previously uncharacterized interactions involving (i) HrpG, a two-component system response regulator responsible for the expression of X. axonopodis pv. citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; (ii) HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV; (iii) HrpB1, HrpD6, and HrpW; and (iv) HrpB2 and HrcU. Homotropic interactions were also identified for the ATPase HrcN. These newly identified protein-protein interactions increase our understanding of the functional integration of phytopathogen-specific type III secretion system components and suggest new hypotheses regarding the molecular mechanisms underlying Xanthomonas pathogenicity. PMID:15342589

  14. Structural Basis of Chaperone Recognition of Type III Secretion System Minor Translocator Proteins*

    PubMed Central

    Job, Viviana; Matteï, Pierre-Jean; Lemaire, David; Attree, Ina; Dessen, Andréa

    2010-01-01

    The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the “translocators”) are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development. PMID:20385547

  15. Structural basis of chaperone recognition of type III secretion system minor translocator proteins.

    PubMed

    Job, Viviana; Matteï, Pierre-Jean; Lemaire, David; Attree, Ina; Dessen, Andréa

    2010-07-23

    The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the "translocators") are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development.

  16. 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.

  17. NMR characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa

    PubMed Central

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

    2017-01-01

    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 homologs 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 CD and 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 residues 16–41 and 55–76. NMR backbone dynamics data show that the helices in PcrG have semi-rigid flexibility and they tumble as a single entity with similar backbone dynamics. NMR titrations show that the entire length of PcrG residues from 9–76 is involved in binding to PcrV. Thus the PcrG family of T3SS chaperone proteins is essentially partially folded. PMID:26451841

  18. The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL

    PubMed Central

    Shaulov, Lihi; Gershberg, Jenia; Deng, Wanyin; Finlay, B. Brett

    2017-01-01

    ABSTRACT The type III secretion system (T3SS) is a multiprotein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. To ensure that effector proteins are efficiently translocated into the host cell, bacteria must be able to sense their contact with the host cell. In this study, we found that EscP, which was previously shown to function as the ruler protein of the enteropathogenic Escherichia coli T3SS, is also involved in the switch from the secretion of translocator proteins to the secretion of effector proteins. In addition, we demonstrated that EscP can interact with the gatekeeper protein SepL and that the EscP-SepL complex dissociates upon a calcium concentration drop. We suggest a model in which bacterial contact with the host cell is accompanied by a drop in the calcium concentration that causes SepL-EscP complex dissociation and triggers the secretion of effector proteins. PMID:28049143

  19. Francisella IglG protein and the DUF4280 proteins: PAAR-like proteins in non-canonical Type VI secretion systems?

    PubMed Central

    Lays, Claire; Tannier, Eric; Henry, Thomas

    2016-01-01

    Type VI secretion systems (T6SS) are bacterial molecular machines translocating effector proteins into target cells. T6SS are widely present in Gram-negative bacteria where they predominantly act to kill neighboring bacteria. This secretion system is reminiscent of the tail of contractile bacteriophages and consists of a contractile sheath anchored in the bacterial envelope and an inner tube made of stacks of the Hcp protein. The Hcp tube is capped with a VgrG trimer and a spike protein termed PAAR, which acts as the membrane-puncturing device. Francisella tularensis, the agent of tularemia, is an intracellular bacterium replicating within the host cytosol. Upon entry into the host cell, F. tularensis rapidly lyses the host vacuolar membrane to reach the host cytosol. This escape is dependent on the Francisella Pathogenicity Island (FPI), which is encoding an atypical T6SS. Among the 17 proteins encoded by the FPI, most of them required for virulence, eight have some homology to canonical T6SS proteins. We recently identified the function of one protein of unknown function encoded within the FPI, IglG. By three-dimensional modelling and following validation by different techniques, we found that IglG adopts a fold resembling the one of PAAR proteins. Importantly, IglG features a domain of unknown function DUF4280, present in numerous bacterial species. We thus propose to rename this domain of unknown function, PAAR-like domain, and discuss here the characteristics of this domain and its distribution in both Gram-negative and Gram-positive bacteria. PMID:28357328

  20. Francisella IglG protein and the DUF4280 proteins: PAAR-like proteins in non-canonical Type VI secretion systems?

    PubMed

    Lays, Claire; Tannier, Eric; Henry, Thomas

    2016-10-29

    Type VI secretion systems (T6SS) are bacterial molecular machines translocating effector proteins into target cells. T6SS are widely present in Gram-negative bacteria where they predominantly act to kill neighboring bacteria. This secretion system is reminiscent of the tail of contractile bacteriophages and consists of a contractile sheath anchored in the bacterial envelope and an inner tube made of stacks of the Hcp protein. The Hcp tube is capped with a VgrG trimer and a spike protein termed PAAR, which acts as the membrane-puncturing device. Francisella tularensis, the agent of tularemia, is an intracellular bacterium replicating within the host cytosol. Upon entry into the host cell, F. tularensis rapidly lyses the host vacuolar membrane to reach the host cytosol. This escape is dependent on the Francisella Pathogenicity Island (FPI), which is encoding an atypical T6SS. Among the 17 proteins encoded by the FPI, most of them required for virulence, eight have some homology to canonical T6SS proteins. We recently identified the function of one protein of unknown function encoded within the FPI, IglG. By three-dimensional modelling and following validation by different techniques, we found that IglG adopts a fold resembling the one of PAAR proteins. Importantly, IglG features a domain of unknown function DUF4280, present in numerous bacterial species. We thus propose to rename this domain of unknown function, PAAR-like domain, and discuss here the characteristics of this domain and its distribution in both Gram-negative and Gram-positive bacteria.

  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

    2016-05-27

    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.

  3. Hcp family proteins secreted via the type VI secretion system coordinately regulate Escherichia coli K1 interaction with human brain microvascular endothelial cells.

    PubMed

    Zhou, Yan; Tao, Jing; Yu, Hao; Ni, Jinjing; Zeng, Lingbing; Teng, Qihui; Kim, Kwang Sik; Zhao, Guo-Ping; Guo, Xiaokui; Yao, Yufeng

    2012-03-01

    Type VI secretion systems (T6SSs) are involved in the pathogenicity of several gram-negative bacteria. Based on sequence analysis, we found that a cluster of Escherichia coli virulence factors (EVF) encoding a putative T6SS exists in the genome of the meningitis-causing E. coli K1 strain RS218. The T6SS-associated deletion mutants exhibited significant defects in binding to and invasion of human brain microvascular endothelial cells (HBMEC) compared with the parent strain. Hcp family proteins (the hallmark of T6SS), including Hcp1 and Hcp2, were localized in the bacterial outer membrane, but the involvements of Hcp1 and Hcp2 have been shown to differ in E. coli-HBMEC interaction. The deletion mutant of hcp2 showed defects in the bacterial binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SS-dependent manner and induced actin cytoskeleton rearrangement, apoptosis, and the release of interleukin-6 (IL-6) and IL-8 in HBMEC. These findings demonstrate that the T6SS is functional in E. coli K1, and two Hcp family proteins participate in different steps of E. coli interaction with HBMEC in a coordinate manner, e.g., binding to and invasion of HBMEC, the cytokine and chemokine release followed by cytoskeleton rearrangement, and apoptosis in HBMEC. This is the first demonstration of the role of T6SS in meningitis-causing E. coli K1, and T6SS-associated Hcp family proteins are likely to contribute to the pathogenesis of E. coli meningitis.

  4. 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.

  5. 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.

  6. BipC, a Predicted Burkholderia pseudomallei Type 3 Secretion System Translocator Protein with Actin Binding Activity.

    PubMed

    Vander Broek, Charles W; Zainal Abidin, Nurhamimah; Stevens, Joanne M

    2017-01-01

    Burkholderia pseudomallei is an intracellular bacterial pathogen and the causative agent of melioidosis, a severe disease of humans and animals. Like other clinically important Gram-negative bacteria, fundamental to B. pseudomallei pathogenesis is the Bsa Type III Secretion System. The Bsa system injects bacterial effector proteins into the cytoplasm of target host cells subverting cellular pathways for the benefit of the bacteria. It is required for invasion of non-phagocytic host cells, escape from the endocytic compartment into the host cell cytoplasm, and for virulence in murine models of melioidosis. We have recently described the repertoire of effector proteins secreted by the B. pseudomallei Bsa system, however the functions of many of these effector proteins remain an enigma. One such protein is BipC, a homolog of the translocator/effector proteins SipC and IpaC from Salmonella spp. and Shigella flexneri respectively. SipC and IpaC each have separate and distinct roles acting both as translocators, involved in creating a pore in the eukaryotic cell membrane through which effector proteins can transit, and as effectors by interacting with and polymerizing host cell actin. In this study, pull-down assays demonstrate an interaction between BipC and actin. Furthermore, we show that BipC directly interacts with actin, preferentially with actin polymers (F-actin) and has the ability to polymerize actin in a similar manner as that described for SipC. Yet unlike SipC, BipC does not stabilize F-actin filaments, indicating a functionally distinct interaction with actin. Expression of Myc-tagged BipC in HeLa cells induces the formation of pseudopodia similar to that seen for IpaC. This study explores the effector function of BipC and reveals that actin interaction is conserved within the BipC/SipC/IpaC family of translocator/effector proteins.

  7. BipC, a Predicted Burkholderia pseudomallei Type 3 Secretion System Translocator Protein with Actin Binding Activity

    PubMed Central

    Vander Broek, Charles W.; Zainal Abidin, Nurhamimah; Stevens, Joanne M.

    2017-01-01

    Burkholderia pseudomallei is an intracellular bacterial pathogen and the causative agent of melioidosis, a severe disease of humans and animals. Like other clinically important Gram-negative bacteria, fundamental to B. pseudomallei pathogenesis is the Bsa Type III Secretion System. The Bsa system injects bacterial effector proteins into the cytoplasm of target host cells subverting cellular pathways for the benefit of the bacteria. It is required for invasion of non-phagocytic host cells, escape from the endocytic compartment into the host cell cytoplasm, and for virulence in murine models of melioidosis. We have recently described the repertoire of effector proteins secreted by the B. pseudomallei Bsa system, however the functions of many of these effector proteins remain an enigma. One such protein is BipC, a homolog of the translocator/effector proteins SipC and IpaC from Salmonella spp. and Shigella flexneri respectively. SipC and IpaC each have separate and distinct roles acting both as translocators, involved in creating a pore in the eukaryotic cell membrane through which effector proteins can transit, and as effectors by interacting with and polymerizing host cell actin. In this study, pull-down assays demonstrate an interaction between BipC and actin. Furthermore, we show that BipC directly interacts with actin, preferentially with actin polymers (F-actin) and has the ability to polymerize actin in a similar manner as that described for SipC. Yet unlike SipC, BipC does not stabilize F-actin filaments, indicating a functionally distinct interaction with actin. Expression of Myc-tagged BipC in HeLa cells induces the formation of pseudopodia similar to that seen for IpaC. This study explores the effector function of BipC and reveals that actin interaction is conserved within the BipC/SipC/IpaC family of translocator/effector proteins. PMID:28770177

  8. An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens.

    PubMed

    Ma, Lay-Sun; Lin, Jer-Sheng; Lai, Erh-Min

    2009-07-01

    An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpL(M), an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL(M) and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of beta-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL(M) is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL(M) mutants with substitutions or deletions in the Walker A motif failed to complement the impL(M) deletion mutant for Hcp secretion, which provided evidence that ImpL(M) may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL(M) and another essential T6SS component, ImpK(L). Topology and biochemical fractionation analyses suggested that ImpK(L) is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL(M)-ImpK(L) interaction domains suggested that ImpL(M) interacts with ImpK(L) via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL(M) interacts with ImpK(L), and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.

  9. Unconventional protein secretion: an evolving mechanism

    PubMed Central

    Malhotra, Vivek

    2013-01-01

    The process by which proteins are secreted without entering the classical endoplasmic reticulum (ER)–Golgi complex pathway, in eukaryotic cells, is conveniently called unconventional protein secretion. Recent studies on one such protein called Acb1 have revealed a number of components involved in its secretion. Interestingly, conditions that promote the secretion of Acb1 trigger the biogenesis of a new compartment called CUPS (Compartment for Unconventional Protein Secretion). CUPS form near the ER exit site but lack ER-specific proteins. Other proteins that share some of the features common with the secretion of Acb1 are interleukin-1β and tissue transglutaminase. Here I will review recent advances made in the field and propose a new model for unconventional protein secretion. PMID:23665917

  10. Molecular characterization and polyclonal antibody generation against core component CagX protein of Helicobacter pylori type IV secretion system

    PubMed Central

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

    2014-01-01

    Gram-negative bacteria Helicobacter pylori cause gastric ulcer, duodenal cancer, and found in almost half of the world’s residents. The protein responsible for this disease is secreted through type IV secretion system (TFSS) of H. pylori. TFSS is encoded by 40-kb region of chromosomal DNA known as cag-pathogenicity island (PAI). TFSS comprises of three major components: cytoplasmic/inner membrane ATPase, transmembrane core-complex and outer membranous pilli, and associated subunits. Core complex consists of CagX, CagT, CagM, and Cag3(δ) proteins as per existing knowledge. In this study, we have characterized one of the important component of core-complex forming sub-unit protein, i.e., CagX. Complete ORF of CagX except signal peptide coding region was cloned and expressed in pET28a vector. Purification of CagX protein was performed, and polyclonal anti-sera against full-length recombinant CagX were raised in rabbit model. We obtained a very specific and high titer, CagX anti-sera that were utilized to characterize endogenous CagX. Surface localization of CagX was also seen by immunofluorescence microscopy. In short for the first time a full-length CagX was characterized, and we showed that CagX is the part of high molecular weight core complex, which is important for assembly and function of H. pylori TFSS. PMID:24637488

  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. Copyright © 2015. Published by Elsevier Ltd.

  12. Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains.

    PubMed

    Kneuper, Holger; Cao, Zhen Ping; Twomey, Kate B; Zoltner, Martin; Jäger, Franziska; Cargill, James S; Chalmers, James; van der Kooi-Pol, Magdalena M; van Dijl, Jan Maarten; Ryan, Robert P; Hunter, William N; Palmer, Tracy

    2014-09-01

    The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.

  13. Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains

    PubMed Central

    Kneuper, Holger; Cao, Zhen Ping; Twomey, Kate B; Zoltner, Martin; Jäger, Franziska; Cargill, James S; Chalmers, James; van der Kooi-Pol, Magdalena M; van Dijl, Jan Maarten; Ryan, Robert P; Hunter, William N; Palmer, Tracy

    2014-01-01

    The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions. PMID:25040609

  14. Antibacterial Activity of the Contact and Complement Systems Is Blocked by SIC, a Protein Secreted by Streptococcus pyogenes*

    PubMed Central

    Frick, Inga-Maria; Shannon, Oonagh; Åkesson, Per; Mörgelin, Matthias; Collin, Mattias; Schmidtchen, Artur; Björck, Lars

    2011-01-01

    Recent studies have shown that activation of complement and contact systems results in the generation of antibacterial peptides. Streptococcus pyogenes, a major bacterial pathogen in humans, exists in >100 different serotypes due to sequence variation in the surface-associated M protein. Cases of invasive and life-threatening S. pyogenes infections are commonly associated with isolates of the M1 serotype, and in contrast to the large majority of M serotypes, M1 isolates all secrete the SIC protein. Here, we show that SIC interferes with the activation of the contact system and blocks the activity of antibacterial peptides generated through complement and contact activation. This effect promotes the growth of S. pyogenes in human plasma, and in a mouse model of S. pyogenes sepsis, SIC enhances bacterial dissemination, results which help explain the high frequency of severe S. pyogenes infections caused by isolates of the M1 serotype. PMID:21068386

  15. 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

  16. Novel protein-protein interactions of the Yersinia pestis type III secretion system elucidated with a matrix analysis by surface plasmon resonance and mass spectrometry.

    PubMed

    Swietnicki, Wieslaw; O'Brien, Sarah; Holman, Kari; Cherry, Scott; Brueggemann, Ernst; Tropea, Joseph E; Hines, Harry B; Waugh, David S; Ulrich, Robert G

    2004-09-10

    Binary complexes formed by components of the Yersinia pestis type III secretion system were investigated by surface plasmon resonance (SPR) and matrix-assisted laser desorption time-of-flight mass spectrometry. Pairwise interactions between 15 recombinant Yersinia outer proteins (Yops), regulators, and chaperones were first identified by SPR. Mass spectrometry confirmed over 80% of the protein-protein interactions suggested by SPR, and new binding partners were further characterized. The Yop secretion protein (Ysc) M2 of Yersinia enterocolitica and LcrQ of Y. pestis, formerly described as ligands only for the specific Yop chaperone (Syc) H, formed stable complexes with SycE. Additional previously unreported complexes of YscE with the translocation regulator protein TyeA and the thermal regulator protein YmoA and multiple potential protein contacts by YscE, YopK, YopH, and LcrH were also identified. Because only stably folded proteins were examined, the interactions we identified are likely to occur either before or after transfer through the injectosome to mammalian host cells and may have relevance to understanding disease processes initiated by the plague bacterium.

  17. Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells

    PubMed Central

    Sakalis, Philippe A; van Heusden, G Paul H; Hooykaas, Paul J J

    2014-01-01

    Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20–25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli. PMID:24376037

  18. Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells.

    PubMed

    Sakalis, Philippe A; van Heusden, G Paul H; Hooykaas, Paul J J

    2014-02-01

    Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20-25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli.

  19. 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

  20. 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

  1. A Rab escort protein integrates the secretion system with TOR signaling and ribosome biogenesis.

    PubMed

    Singh, Jaspal; Tyers, Mike

    2009-08-15

    The coupling of environmental conditions to cell growth and division is integral to cell fitness. In Saccharomyces cerevisiae, the transcription factor Sfp1 couples nutrient status to cell growth rate by controlling the expression of ribosome biogenesis (Ribi) and ribosomal protein (RP) genes. Sfp1 is localized to the nucleus in rich nutrients, but upon nutrient limitation or target of rapamycin (TOR) pathway inhibition by rapamycin, Sfp1 rapidly exits the nucleus, leading to repression of the Ribi/RP regulons. Through systematic cell-based screens we found that many components of the secretory system influence Sfp1 localization. Notably, the essential Rab escort protein Mrs6 exhibited a nutrient-sensitive interaction with Sfp1. Overexpression of Mrs6 prevented nuclear localization of Sfp1 in rich nutrients, whereas loss of Mrs6 resulted in nuclear Sfp1 localization in poor nutrients. These effects were specific to Sfp1 and independent of the protein kinase C (PKC) pathway, suggesting that Mrs6 lies in a distinct branch of TOR and ribosome biogenesis regulation. Rapamycin-resistant alleles of MRS6 were defective in the cytoplasmic retention of Sfp1, the control of cell size, and in the repression of the Ribi/RP regulons. The Sfp1-Mrs6 interaction is a nexus for growth regulation that links the secretory system and TOR-dependent nutrient signaling to ribosome biogenesis.

  2. A Rab escort protein integrates the secretion system with TOR signaling and ribosome biogenesis

    PubMed Central

    Singh, Jaspal; Tyers, Mike

    2009-01-01

    The coupling of environmental conditions to cell growth and division is integral to cell fitness. In Saccharomyces cerevisiae, the transcription factor Sfp1 couples nutrient status to cell growth rate by controlling the expression of ribosome biogenesis (Ribi) and ribosomal protein (RP) genes. Sfp1 is localized to the nucleus in rich nutrients, but upon nutrient limitation or target of rapamycin (TOR) pathway inhibition by rapamycin, Sfp1 rapidly exits the nucleus, leading to repression of the Ribi/RP regulons. Through systematic cell-based screens we found that many components of the secretory system influence Sfp1 localization. Notably, the essential Rab escort protein Mrs6 exhibited a nutrient-sensitive interaction with Sfp1. Overexpression of Mrs6 prevented nuclear localization of Sfp1 in rich nutrients, whereas loss of Mrs6 resulted in nuclear Sfp1 localization in poor nutrients. These effects were specific to Sfp1 and independent of the protein kinase C (PKC) pathway, suggesting that Mrs6 lies in a distinct branch of TOR and ribosome biogenesis regulation. Rapamycin-resistant alleles of MRS6 were defective in the cytoplasmic retention of Sfp1, the control of cell size, and in the repression of the Ribi/RP regulons. The Sfp1–Mrs6 interaction is a nexus for growth regulation that links the secretory system and TOR-dependent nutrient signaling to ribosome biogenesis. PMID:19684114

  3. Identification of secreted bacterial proteins by noncanonical amino acid tagging.

    PubMed

    Mahdavi, Alborz; Szychowski, Janek; Ngo, John T; Sweredoski, Michael J; Graham, Robert L J; Hess, Sonja; Schneewind, Olaf; Mazmanian, Sarkis K; Tirrell, David A

    2014-01-07

    Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.

  4. The inner rod protein controls substrate switching and needle length in a Salmonella type III secretion system.

    PubMed

    Lefebre, Matthew D; Galán, Jorge E

    2014-01-14

    Type III secretion machines are essential for the biology of many bacteria that are pathogenic or symbiotic for animals, plants, or insects. They exert their function by delivering bacterial effector proteins into target eukaryotic cells. The core component of these machines is the needle complex, a multiprotein structure that spans the bacterial envelope and serves as a conduit for proteins that transit this secretion pathway. The needle complex is composed of a multiring base embedded in the bacterial envelope and a filament-like structure, the needle, that projects from the bacterial surface and is linked to the base by the inner rod. Assembly of the needle complex proceeds in a step-wise fashion that is initiated by the assembly of the base and is followed by the export of the building subunits for the needle and inner rod substructures. Once assembled, the needle complex reprograms its specificity and becomes competent for the secretion of effector proteins. Here through genetic, biochemical, and electron microscopy analyses of the Salmonella inner rod protein subunit PrgJ we present evidence that the assembly of the inner rod dictates the timing of substrate switching and needle length. Furthermore, the identification of mutations in PrgJ that specifically alter the hierarchy of protein secretion provides additional support for a complex role of the inner rod substructure in type III secretion.

  5. 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.

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

    PubMed Central

    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

    2013-01-01

    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 dr1245 gene 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. PMID:23441204

  7. Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion.

    PubMed

    Ling Lin Fu; Zi Rong Xu; Wei Fen Li; Jiang Bing Shuai; Ping Lu; Chun Xia Hu

    2007-01-01

    The absence of an outer membrane in Bacillus subtilis can simplify the protein secretion pathways and allow the organism to secrete high levels of extracellular proteins. Of the three known secretory routes, Sec-SRP pathway can direct the majority of secretory proteins into the growth medium. Alternatively, a small number of exoproteins with specific functions are secreted via Tat pathway or ABC transporters in B. subtilis. The discriminating function of precursor proteins among these pathways is largely attributed to the distinct structure of their cleavable signal peptides. Individual secretion machinery components with their special functions are involved in the total flow of proteins from the cytoplasm to the medium. Notably, multiple regulators with signal transduction functions can affect expression of secretion machinery as well as their post-transcriptional actions for protein secretion, resulting in the complicated networks in B. subtilis. Ultimately, according to the available knowledge of secretion machinery, several approaches aimed at optimizing protein secretion are discussed.

  8. 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

  9. Toxins and Secretion Systems of Photorhabdus luminescens

    PubMed Central

    Rodou, Athina; Ankrah, Dennis O.; Stathopoulos, Christos

    2010-01-01

    Photorhabdus luminescens is a nematode-symbiotic, gram negative, bioluminescent bacterium, belonging to the family of Enterobacteriaceae. Recent studies show the importance of this bacterium as an alternative source of insecticides, as well as an emerging human pathogen. Various toxins have been identified and characterized in this bacterium. These toxins are classified into four major groups: the toxin complexes (Tcs), the Photorhabdus insect related (Pir) proteins, the “makes caterpillars floppy” (Mcf) toxins and the Photorhabdus virulence cassettes (PVC); the mechanisms however of toxin secretion are not fully elucidated. Using bioinformatics analysis and comparison against the components of known secretion systems, multiple copies of components of all known secretion systems, except the ones composing a type IV secretion system, were identified throughout the entire genome of the bacterium. This indicates that Photorhabdus luminescens has all the necessary means for the secretion of virulence factors, thus it is capable of establishing a microbial infection. PMID:22069636

  10. Toxins and secretion systems of Photorhabdus luminescens.

    PubMed

    Rodou, Athina; Ankrah, Dennis O; Stathopoulos, Christos

    2010-06-01

    Photorhabdus luminescens is a nematode-symbiotic, gram negative, bioluminescent bacterium, belonging to the family of Enterobacteriaceae. Recent studies show the importance of this bacterium as an alternative source of insecticides, as well as an emerging human pathogen. Various toxins have been identified and characterized in this bacterium. These toxins are classified into four major groups: the toxin complexes (Tcs), the Photorhabdus insect related (Pir) proteins, the "makes caterpillars floppy" (Mcf) toxins and the Photorhabdus virulence cassettes (PVC); the mechanisms however of toxin secretion are not fully elucidated. Using bioinformatics analysis and comparison against the components of known secretion systems, multiple copies of components of all known secretion systems, except the ones composing a type IV secretion system, were identified throughout the entire genome of the bacterium. This indicates that Photorhabdus luminescens has all the necessary means for the secretion of virulence factors, thus it is capable of establishing a microbial infection.

  11. Structure of an Essential Type IV Pilus Biogenesis Protein Provides Insights into Pilus and Type II Secretion Systems

    PubMed Central

    Yamagata, Atsushi; Milgotina, Ekaterina; Scanlon, Karen; Craig, Lisa; Tainer, John A.; Donnenberg, Michael S.

    2012-01-01

    Type IV pili (T4Ps) are long cell surface filaments, essential for microcolony formation, tissue adherence, motility, transformation, and virulence by human pathogens. The enteropathogenic E. colibundle-forming pilus (BFP) is a prototypic T4P assembled and powered by BfpD, a conserved GspE secretion superfamily ATPase held by inner membrane proteins BfpC andBfpE, a GspF-family membrane protein. Although the T4P assembly machinery shares similarity with type II secretion (T2S) systems, the structural biochemistry of the T4P machine has been obscure. Here, we report the crystal structure of the two-domain BfpC cytoplasmic region (N-BfpC), responsible for binding to ATPase BfpD and membrane protein BfpE. The N-BfpC structure reveals a prominent central cleft between two α/β domains. Despite negligible sequence similarity, N-BfpC resembles PilM, a cytoplasmic T4P biogenesis protein.Yet surprisingly, N-BfpC has far greaterstructural similarity to T2S component EpsL, with which it also shares virtually no sequence identity. The C-terminus of the cytoplasmic domain, which leads to the transmembrane segment not present in the crystal structure, exits N-BfpC at a positively-charged surface that most likely interacts with the inner membrane, positioning its central cleft for interactions with other Bfp components.Point mutations in surface-exposed N-BfpC residues predicted to be critical for interactions among BfpC, BfpE and BfpD disrupt pilus biogenesis without precluding interactions with BfpE and BfpD and without affecting BfpD ATPase activity. These results illuminate the relationships between T4P biogenesis and T2S systems,imply that subtle changes in component residue interactions can have profound effects on function and pathogenesis, and suggest that T4P systems may be disrupted by inhibitors that donot preclude component assembly. PMID:22387466

  12. 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 to Bacteroides 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 for B. fragilis strains 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 species B. fragilis are 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.

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

    PubMed Central

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

    2016-01-01

    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 to Bacteroides 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 for B. fragilis strains 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 species B. fragilis are 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

  14. 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

  15. Purification and Characteriztion of the Type III Secretion System Protein from Burkholderia mallei

    DTIC Science & Technology

    2013-08-01

    maltose -binding protein (MBP) fusion at the amino (N)-terminus. 2.2 Protein Expression The construct was transformed into E. coli T7 Express...performance (HP) column (GE Healthcare) and eluted with a linear gradient of 10 mM maltose . The fractions containing the protein were pooled, the...MBPTrap HP (GE Healthcare) affinity column and eluted with a linear gradient of 10 mM of maltose . 4 Figure 2. SDS-PAGE results for purified BsaS

  16. Ehrlichia chaffeensis Tandem Repeat Proteins and Ank200 are Type 1 Secretion System Substrates Related to the Repeats-in-Toxin Exoprotein Family

    PubMed Central

    Wakeel, Abdul; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; McBride, Jere W.

    2011-01-01

    Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host–pathogen interactions including DNA binding and a network of protein–protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E. chaffeensis TRP47, TRP32, TRP120, and Ank200 were not secreted in the Agrobacterium tumefaciens Cre recombinase reporter assay routinely used to identify T4SS substrates. In contrast, all TRPs and the Ank200 proteins were secreted by the Escherichia coli complemented with the hemolysin secretion system (T1SS), and secretion was reduced in a T1SS mutant (ΔTolC), demonstrating that these proteins are T1SS substrates. Moreover, T1SS secretion signals were identified in the C-terminal domains of the TRPs and Ank200, and a detailed bioinformatic analysis of E. chaffeensis TRPs and Ank200 revealed features consistent with those described in the repeats-in-toxins (RTX) family of exoproteins, including glycine- and aspartate-rich tandem repeats, homology with ATP-transporters, a non-cleavable C-terminal T1SS signal, acidic pIs, and functions consistent with other T1SS substrates. Using a heterologous E. coli T1SS, this investigation has identified the first Ehrlichia T1SS substrates supporting the conclusion that the T1SS and corresponding substrates are involved in molecular host–pathogen interactions that contribute to Ehrlichia pathobiology. Further investigation of the relationship between Ehrlichia TRPs, Ank200, and the RTX exoprotein family may lead to a greater understanding of the importance of T1SS substrates and specific functions of T1SS in the pathobiology of obligately intracellular bacteria. PMID:22919588

  17. 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

  18. 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

  19. 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.

  20. Characterization of Small-Molecule Scaffolds That Bind to the Shigella Type III Secretion System Protein IpaD.

    PubMed

    Dey, Supratim; Anbanandam, Asokan; Mumford, Ben E; De Guzman, Roberto N

    2017-09-21

    Many pathogens such as Shigella and other bacteria assemble the type III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salt sterols. In this study we identified four new small-molecule scaffolds that bind to IpaD, based on the methylquinoline, pyrrolidine-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small-molecule inhibitors of IpaD that could lead to new anti-infectives. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Novel Protein-Protein Interactions of the Yersinia pestis Type III Secretion System Elucidated With a Matrix Analysis by Surface Plasmon Resonance and Mass Spectrometry

    DTIC Science & Technology

    2004-09-10

    desorption time-of-flight (MALDI-TOF) mass spectrometry. Pair-wise interactions between fifteen recombinant Yersinia outer proteins ( Yops ), regulators...binding partners were further characterized. The Yop secretion protein (Ysc) M2 of Y. enterocolitica and LcrQ of Y. pestis, formerly described as ligands... proteins ( Yops ), regulators and chaperones were first identified by SPR. Mass spectrometry confirmed over 80% of the protein - protein interactions

  2. Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

    PubMed Central

    2012-01-01

    Summary: Flagellar and translocation-associated type III secretion (T3S) systems are present in most Gram-negative plant- and animal-pathogenic bacteria and are often essential for bacterial motility or pathogenicity. The architectures of the complex membrane-spanning secretion apparatuses of both systems are similar, but they are associated with different extracellular appendages, including the flagellar hook and filament or the needle/pilus structures of translocation-associated T3S systems. The needle/pilus is connected to a bacterial translocon that is inserted into the host plasma membrane and mediates the transkingdom transport of bacterial effector proteins into eukaryotic cells. During the last 3 to 5 years, significant progress has been made in the characterization of membrane-associated core components and extracellular structures of T3S systems. Furthermore, transcriptional and posttranscriptional regulators that control T3S gene expression and substrate specificity have been described. Given the architecture of the T3S system, it is assumed that extracellular components of the secretion apparatus are secreted prior to effector proteins, suggesting that there is a hierarchy in T3S. The aim of this review is to summarize our current knowledge of T3S system components and associated control proteins from both plant- and animal-pathogenic bacteria. PMID:22688814

  3. Secretory fluorescent protein, a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system.

    PubMed

    Teng, Chao-Yi; Wu, Tzong-Yuan

    2007-07-01

    The advantages of using traceable fluorescent protein (enhanced green fluorescent protein; EGFP) and a secretory alkaline phosphatase (SEAP) have been used to generate a reporter gene: the secretory fluorescent protein (SEFP). Sf21 cells, infected with the recombinant baculovirus containing the SEFP gene, revealed both traceable fluorescence and easily detectable alkaline phosphatase activity in the culture medium. The distribution of SEFP within the cells revealed that it was excluded from the nucleus, implying that the accumulation of SEFP in a secretory pathway, similar to that of the secretion signal-tagged FPs. Furthermore, the time- and dose-dependent release from the blockage of brefeldin A (BFA) confirmed that the secretion of SEFP was mediated by the secretion pathway and excluded leakage from viral infection. This SEFP reporter gene with traceable fluorescence and alkaline phosphatase activity may become a useful tool for studies on secretory protein production.

  4. Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae.

    PubMed

    Hou, Jin; Tyo, Keith E J; Liu, Zihe; Petranovic, Dina; Nielsen, Jens

    2012-08-01

    The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals, and it is also provides a platform for the production of many heterologous proteins of medical or industrial interest. Therefore, many studies have focused on metabolic engineering S. cerevisiae to improve the recombinant protein production, and with the development of systems biology, it is interesting to see how this approach can be applied both to gain further insight into protein production and secretion and to further engineer the cell for improved production of valuable proteins. In this review, the protein post-translational modification such as folding, trafficking, and secretion, steps that are traditionally studied in isolation will here be described in the context of the whole system of protein secretion. Furthermore, examples of engineering secretion pathways, high-throughput screening and systems biology applications of studying protein production and secretion are also given to show how the protein production can be improved by different approaches. The objective of the review is to describe individual biological processes in the context of the larger, complex protein synthesis network.

  5. The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation

    PubMed Central

    Hausner, Jens; Hartmann, Nadine; Jordan, Michael

    2016-01-01

    ABSTRACT The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which spans both bacterial membranes and translocates effector proteins into plant cells. The assembly of the T3S system presumably involves the predicted lytic transglycosylase (LT) HpaH, which is encoded adjacent to the T3S gene cluster. Bacterial LTs degrade peptidoglycan and often promote the formation of membrane-spanning macromolecular protein complexes. In the present study, we show that HpaH localizes to the bacterial periplasm and binds to peptidoglycan as well as to components of the T3S system, including the predicted periplasmic inner rod proteins HrpB1 and HrpB2 as well as the pilus protein HrpE. In vivo translocation assays revealed that HpaH promotes the translocation of various effector proteins and of early substrates of the T3S system, suggesting a general contribution of HpaH to type III-dependent protein export. Mutant studies and the analysis of reporter fusions showed that the N-terminal region of HpaH contributes to protein function and is proteolytically cleaved. The N-terminally truncated HpaH cleavage product is secreted into the extracellular milieu by a yet-unknown transport pathway, which is independent of the T3S system. PMID:27895129

  6. The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation.

    PubMed

    Hausner, Jens; Hartmann, Nadine; Jordan, Michael; Büttner, Daniela

    2017-02-01

    The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which spans both bacterial membranes and translocates effector proteins into plant cells. The assembly of the T3S system presumably involves the predicted lytic transglycosylase (LT) HpaH, which is encoded adjacent to the T3S gene cluster. Bacterial LTs degrade peptidoglycan and often promote the formation of membrane-spanning macromolecular protein complexes. In the present study, we show that HpaH localizes to the bacterial periplasm and binds to peptidoglycan as well as to components of the T3S system, including the predicted periplasmic inner rod proteins HrpB1 and HrpB2 as well as the pilus protein HrpE. In vivo translocation assays revealed that HpaH promotes the translocation of various effector proteins and of early substrates of the T3S system, suggesting a general contribution of HpaH to type III-dependent protein export. Mutant studies and the analysis of reporter fusions showed that the N-terminal region of HpaH contributes to protein function and is proteolytically cleaved. The N-terminally truncated HpaH cleavage product is secreted into the extracellular milieu by a yet-unknown transport pathway, which is independent of the T3S system. Copyright © 2017 American Society for Microbiology.

  7. Milk secretion: The role of SNARE proteins.

    PubMed

    Truchet, Sandrine; Chat, Sophie; Ollivier-Bousquet, Michèle

    2014-03-01

    During lactation, polarized mammary epithelial secretory cells (MESCs) secrete huge quantities of the nutrient molecules that make up milk, i.e. proteins, fat globules and soluble components such as lactose and minerals. Some of these nutrients are only produced by the MESCs themselves, while others are to a great extent transferred from the blood. MESCs can thus be seen as a crossroads for both the uptake and the secretion with cross-talks between intracellular compartments that enable spatial and temporal coordination of the secretion of the milk constituents. Although the physiology of lactation is well understood, the molecular mechanisms underlying the secretion of milk components remain incompletely characterized. Major milk proteins, namely caseins, are secreted by exocytosis, while the milk fat globules are released by budding, being enwrapped by the apical plasma membrane. Prolactin, which stimulates the transcription of casein genes, also induces the production of arachidonic acid, leading to accelerated casein transport and/or secretion. Because of their ability to form complexes that bridge two membranes and promote their fusion, SNARE (Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor) proteins are involved in almost all intracellular trafficking steps and exocytosis. As SNAREs can bind arachidonic acid, they could be the effectors of the secretagogue effect of prolactin in MESCs. Indeed, some SNAREs have been observed between secretory vesicles and lipid droplets suggesting that these proteins could not only orchestrate the intracellular trafficking of milk components but also act as key regulators for both the coupling and coordination of milk product secretion in response to hormones.

  8. PAAR-Rhs proteins harbor various C-terminal toxins to diversify the antibacterial pathways of type VI secretion systems.

    PubMed

    Ma, Jiale; Sun, Min; Dong, Wenyang; Pan, Zihao; Lu, Chengping; Yao, Huochun

    2017-01-01

    The type VI secretion system (T6SS) of bacteria plays a key role in competing for specific niches by the contact-dependent killing of competitors. Recently, Rhs proteins with polymorphic C-terminal toxin-domains that inhibit or kill neighboring cells were identified. In this report, we identified a novel Rhs with an MPTase4 (Metallopeptidase-4) domain (designated as Rhs-CT1) that showed an antibacterial effect via T6SS in Escherichia coli. We managed to develop a specific strategy by matching the diagnostic domain-architecture of Rhs-CT1 (Rhs with an N-terminal PAAR-motif and a C-terminal toxin domain) for effector retrieval and discovered a series of Rhs-CTs in E. coli. Indeed, the screened Rhs-CT3 with a REase-3 (Restriction endonuclease-3) domain also mediated interbacterial antagonism. Further analysis revealed that vgrGO1 and eagR/DUF1795 (upstream of rhs-ct) were required for the delivery of Rhs-CTs, suggesting eagR as a potential T6SS chaperone. In addition to chaperoned Rhs-CTs, neighborless Rhs-CTs could be classified into a distinct family (Rhs-Nb) sharing close evolutionary relationship with T6SS2-Rhs (encoded in the T6SS2 cluster of E. coli). Notably, the Rhs-Nb-CT5 was confirmed bioinformatically and experimentally to mediate interbacterial antagonism via Hcp2B-VgrG2 module. In a further retrieval analysis, we discovered various toxin/immunity pairs in extensive bacterial species that could be systematically classified into eight referential clans, suggesting that Rhs-CTs greatly diversify the antibacterial pathways of T6SS.

  9. Proteolytic events in the processing of secreted proteins in fungi.

    PubMed

    Calmels, T P; Martin, F; Durand, H; Tiraby, G

    1991-01-01

    Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.

  10. Mutations in the Yersinia pseudotuberculosis Type III Secretion System Needle Protein, YscF, That Specifically Abrogate Effector Translocation into Host Cells▿ †

    PubMed Central

    Davis, Alison J.; Mecsas, Joan

    2007-01-01

    The trafficking of effectors, termed Yops, from Yersinia spp. into host cells is a multistep process that requires the type III secretion system (TTSS). The TTSS has three main structural parts: a base, a needle, and a translocon, which work together to ensure the polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. To understand the interactions that take place at the interface between the tip of the TTSS needle and the translocon, we developed a screen to identify mutations in the needle protein YscF that separated its function in secretion from its role in translocation. We identified 25 translocation-defective (TD) yscF mutants, which fall into five phenotypic classes. Some classes exhibit aberrant needle structure and/or reduced levels of Yop secretion, consistent with known functions for YscF. Strikingly, two yscF TD classes formed needles and secreted Yops normally but displayed distinct translocation defects. Class I yscF TD mutants showed diminished pore formation, suggesting incomplete pore insertion and/or assembly. Class II yscF TD mutants formed pores but showed nonpolar translocation, suggesting unstable needle-translocon interactions. These results indicate that YscF functions in Yop secretion and translocation can be genetically separated. Furthermore, the identification of YscF residues that are required for the assembly of the translocon and/or productive interactions with the translocon has allowed us to initiate the mapping of the needle-translocon interface. PMID:17071752

  11. Mutations in the Yersinia pseudotuberculosis type III secretion system needle protein, YscF, that specifically abrogate effector translocation into host cells.

    PubMed

    Davis, Alison J; Mecsas, Joan

    2007-01-01

    The trafficking of effectors, termed Yops, from Yersinia spp. into host cells is a multistep process that requires the type III secretion system (TTSS). The TTSS has three main structural parts: a base, a needle, and a translocon, which work together to ensure the polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. To understand the interactions that take place at the interface between the tip of the TTSS needle and the translocon, we developed a screen to identify mutations in the needle protein YscF that separated its function in secretion from its role in translocation. We identified 25 translocation-defective (TD) yscF mutants, which fall into five phenotypic classes. Some classes exhibit aberrant needle structure and/or reduced levels of Yop secretion, consistent with known functions for YscF. Strikingly, two yscF TD classes formed needles and secreted Yops normally but displayed distinct translocation defects. Class I yscF TD mutants showed diminished pore formation, suggesting incomplete pore insertion and/or assembly. Class II yscF TD mutants formed pores but showed nonpolar translocation, suggesting unstable needle-translocon interactions. These results indicate that YscF functions in Yop secretion and translocation can be genetically separated. Furthermore, the identification of YscF residues that are required for the assembly of the translocon and/or productive interactions with the translocon has allowed us to initiate the mapping of the needle-translocon interface.

  12. Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome, global protein secretion, and T cells.

    PubMed

    Nyman, Tuula A; Lorey, Martina B; Cypryk, Wojciech; Matikainen, Sampsa

    2017-05-01

    The immune system is our defense system against microbial infections and tissue injury, and understanding how it works in detail is essential for developing drugs for different diseases. Mass spectrometry-based proteomics can provide in-depth information on the molecular mechanisms involved in immune responses. Areas covered: Summarized are the key immunology findings obtained with MS-based proteomics in the past five years, with a focus on inflammasome activation, global protein secretion, mucosal immunology, immunopeptidome and T cells. Special focus is on extracellular vesicle-mediated protein secretion and its role in immune responses. Expert commentary: Proteomics is an essential part of modern omics-scale immunology research. To date, MS-based proteomics has been used in immunology to study protein expression levels, their subcellular localization, secretion, post-translational modifications, and interactions in immune cells upon activation by different stimuli. These studies have made major contributions to understanding the molecular mechanisms involved in innate and adaptive immune responses. New developments in proteomics offer constantly novel possibilities for exploring the immune system. Examples of these techniques include mass cytometry and different MS-based imaging approaches which can be widely used in immunology.

  13. Detection of Secretory Immunoglobulin A in Human Colostrum as Mucosal Immune Response against Proteins of the Type Three Secretion System of Salmonella, Shigella and Enteropathogenic Escherichia Coli

    PubMed Central

    Durand, David; Ochoa, Theresa J.; Bellomo, Sicilia M. E.; Contreras, Carmen A.; Bustamante, Víctor H.; Ruiz, Joaquim; Cleary, Thomas G.

    2013-01-01

    Background Some enteropathogens use the type three secretion system (T3SS) to secrete proteins that allows them to interact with enterocytes and promote bacterial attachment or intracellular survival. These proteins are Salmonella invasion proteins (Sip), invasion plasmid antigens (Ipa) of Shigella and E. coli secreted proteins (Esp) of enteropathogenic E. coli (EPEC). There are no previous studies defining the presence of colostral sIgA against all these three major enteric pathogens. Objective To evaluate the presence of sIgA in colostrum against proteins of the T3SS of Salmonella, Shigella and EPEC. Methods We collected 76 colostrum samples from puerperal women in Lima, Peru. These samples were reacted with T3SS proteins extracted from bacterial culture supernatants and evaluated by Western Blot. Results Antibodies were detected against Salmonella antigens SipA in 75 samples (99%), SipC in 62 (82%) and SipB in 31 (41%); against Shigella antigens IpaC in 70 (92%), IpaB in 68 (89%), IpaA in 66 (87%) and IpaD in 41 (54%); and against EPEC EspC in 70 (92%), EspB-D in 65 (86%) and EspA in 41 (54%). 10% of samples had antibodies against all proteins evaluated; and 42% against all except one protein. There was no sample negative to all these proteins. Conclusions The extraordinarily high frequency of antibodies in colostrum of puerperal women detected in this study against these multiple enteric pathogens, shows evidence of immunological memory and prior exposure to these pathogens, in addition to its possible protective role against infection. PMID:23538526

  14. The Antitoxin Protein of a Toxin-Antitoxin System from Xylella fastidiosa Is Secreted via Outer Membrane Vesicles

    PubMed Central

    Santiago, André da Silva; Mendes, Juliano S.; dos Santos, Clelton A.; de Toledo, Marcelo A. S.; Beloti, Lilian L.; Crucello, Aline; Horta, Maria A. C.; Favaro, Marianna T. de Pinho; Munar, Duber M. M.; de Souza, Alessandra A.; Cotta, Mônica A.; de Souza, Anete P.

    2016-01-01

    The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible

  15. Purification of secreted recombinant proteins from Escherichia coli.

    PubMed

    Le, H V; Trotta, P P

    1991-01-01

    Secretion systems engineered for the expression of heterologous protein in E. coli provide several advantages for subsequent isolation of purified product. Proteins released from the periplasmic space, which represent a small fraction (i.e., 4-10%) of total cell protein, can readily be separated from other cellular proteins by centrifugation of the remaining cellular debris or cross-flow ultrafiltration. The starting material derived from secretion systems is generally of higher purity than comparable material produced from strains expressing cytoplasmically for systems exhibiting similar expression levels. The available evidence suggests that recombinant proteins derived from the periplasm are generally, but not always (44-46), soluble in a nonaggregated form. Consequently, simple purification protocols can be effectively employed for producing homogeneous product with a high yield. The majority of the secreted recombinant proteins reviewed in this chapter were purified by simple one- or two-step chromatography procedures. High-resolution techniques such as reversed phase HPLC were found necessary only in cases where the secreted polypeptides were contaminated with proteolytic degradation variants, e.g., hirudin (51) and beta-endorphin (22). The fact that a high level of biological activity has been shown to be characteristic of purified recombinant proteins secreted into the periplasmic space suggests the presence of a native conformation stabilized by the expected disulfide linkages. Intramolecular disulfide bonds most probably form either as the polypeptide is translocated through the cytoplasmic membrane into the periplasm or within the periplasmic compartment, which has a higher oxidation potential than that found in the cytoplasm (57). Studies performed with hGH (31) and muIL-2 (35) provide excellent examples of differences observed in protein folding and disulfide bond formation between heterologous proteins expressed in the cytoplasmic and periplasmic

  16. Systemic autoimmunity and defective Fas ligand secretion in the absence of the Wiskott-Aldrich syndrome protein

    PubMed Central

    Nikolov, Nikolay P.; Shimizu, Masaki; Cleland, Sophia; Bailey, Daniel; Aoki, Joseph; Strom, Ted; Schwartzberg, Pamela L.; Candotti, Fabio

    2010-01-01

    Autoimmunity is a surprisingly common complication of primary immunodeficiencies, yet the molecular mechanisms underlying this clinical observation are not well understood. One widely known example is provided by Wiskott-Aldrich syndrome (WAS), an X-linked primary immunodeficiency disorder caused by mutations in the gene encoding the WAS protein (WASp) with a high incidence of autoimmunity in affected patients. WASp deficiency affects T-cell antigen receptor (TCR) signaling and T-cell cytokine production, but its role in TCR-induced apoptosis, one of the mechanisms of peripheral immunologic tolerance, has not been investigated. We find that WASp-deficient mice produce autoantibodies and develop proliferative glomerulonephritis with immune complex deposition as they age. We also find that CD4+ T lymphocytes from WASp-deficient mice undergo reduced apoptosis after restimulation through the TCR. While Fas-induced cell death is normal, WASp deficiency affects TCR-induced secretion of Fas ligand (FasL) and other components of secretory granules by CD4+ T cells. These results describe a novel role of WASp in regulating TCR-induced apoptosis and FasL secretion and suggest that WASp-deficient mice provide a good model for the study of autoimmune manifestations of WAS and the development of more specific therapies for these complications. PMID:20457871

  17. The EAL-domain protein FcsR regulates flagella, chemotaxis and type III secretion system in Pseudomonas aeruginosa by a phosphodiesterase independent mechanism.

    PubMed

    Rossello, Jessica; Lima, Analía; Gil, Magdalena; Rodríguez Duarte, Jorge; Correa, Agustín; Carvalho, Paulo C; Kierbel, Arlinet; Durán, Rosario

    2017-08-31

    The second messenger c-di-GMP regulates the switch between motile and sessile bacterial lifestyles. A general feature of c-di-GMP metabolism is the presence of a surprisingly large number of genes coding for diguanylate cyclases and phosphodiesterases, the enzymes responsible for its synthesis and degradation respectively. However, the physiological relevance of this apparent redundancy is not clear, emphasizing the need for investigating the functions of each of these enzymes. Here we focused on the phosphodiesterase PA2133 from Pseudomonas aeruginosa, an important opportunistic pathogen. We phenotypically characterized P. aeruginosa strain K overexpressing PA2133 or its inactive mutant. We showed that biofilm formation and motility are severely impaired by overexpression of PA2133. Our quantitative proteomic approach applied to the membrane and exoprotein fractions revealed that proteins involved in three processes were mostly affected: flagellar motility, type III secretion system and chemotaxis. While inhibition of biofilm formation can be ascribed to the phosphodiesterase activity of PA2133, down-regulation of flagellar, chemotaxis, and type III secretion system proteins is independent of this enzymatic activity. Based on these unexpected effects of PA2133, we propose to rename this gene product FcsR, for Flagellar, chemotaxis and type III secretion system Regulator.

  18. Milk protein concentrations in galactorrhoeic mammary secretions.

    PubMed

    Yap, P L; Pryde, E A; McClelland, D B

    1980-02-01

    Milk protein concentrations were determined either by double antibody radioimmunoassay (IgA) or single radial immunodiffusion (IgG, lactoferrin, lysozyme and albumin) in the mammayr secretions of one nulliparous and three parous female patients with galactorrhoea due to hyperprolactinaemia. Concentrations of all the proteins studied were found to be similar to the concentrations observed in post-partum colostrum. In particular, secretory IgA was the only form of IgA detected in galactorrhoeic secretions. It is suggested that hyperprolactinaemia alone can result in increased mammary synthesis of the milk proteins since the steroid changes associated with a full-term pregnancy and delivery of the placenta did not immediately precede the galactorrhoea in three of the four patients studied.

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

    PubMed

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

    2007-09-25

    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.

  20. A switchable yeast display/secretion system

    PubMed Central

    Van Deventer, James A.; Kelly, Ryan L.; Rajan, Saravanan; Wittrup, K. Dane; Sidhu, Sachdev S.

    2015-01-01

    Display technologies such as yeast and phage display offer powerful alternatives to traditional immunization-based antibody discovery, but require conversion of displayed proteins into soluble form prior to downstream characterization. Here we utilize amber suppression to implement a yeast-based switchable display/secretion system that enables the immediate production of soluble, antibody-like reagents at the end of screening efforts. Model selections in the switchable format remain efficient, and library screening in the switchable format yields renewable sources of affinity reagents exhibiting nanomolar binding affinities. These results confirm that this system provides a seamless link between display-based screening and the production and evaluation of soluble forms of candidate binding proteins. Switchable display/secretion libraries provide a cloning-free, accessible approach to affinity reagent generation. PMID:26333274

  1. A switchable yeast display/secretion system.

    PubMed

    Van Deventer, James A; Kelly, Ryan L; Rajan, Saravanan; Wittrup, K Dane; Sidhu, Sachdev S

    2015-10-01

    Display technologies such as yeast and phage display offer powerful alternatives to traditional immunization-based antibody discovery, but require conversion of displayed proteins into soluble form prior to downstream characterization. Here we utilize amber suppression to implement a yeast-based switchable display/secretion system that enables the immediate production of soluble, antibody-like reagents at the end of screening efforts. Model selections in the switchable format remain efficient, and library screening in the switchable format yields renewable sources of affinity reagents exhibiting nanomolar binding affinities. These results confirm that this system provides a seamless link between display-based screening and the production and evaluation of soluble forms of candidate binding proteins. Switchable display/secretion libraries provide a cloning-free, accessible approach to affinity reagent generation.

  2. The Type VI secretion system spike protein VgrG5 mediates membrane fusion during intercellular spread by pseudomallei group Burkholderia species.

    PubMed

    Toesca, Isabelle J; French, Christopher T; Miller, Jeff F

    2014-04-01

    Pseudomallei group Burkholderia species are facultative intracellular parasites that spread efficiently from cell to cell by a mechanism involving the fusion of adjacent cell membranes. Intercellular fusion requires the function of the cluster 5 type VI secretion system (T6SS-5) and its associated valine-glycine repeat protein, VgrG5. Here we show that VgrG5 alleles are conserved and functionally interchangeable between Burkholderia pseudomallei and its relatives B. mallei, B. oklahomensis, and B. thailandensis. We also demonstrate that the integrity of the VgrG5 C-terminal domain is required for fusogenic activity, and we identify sequence motifs, including two hydrophobic segments, that are important for fusion. Mutagenesis and secretion experiments using B. pseudomallei strains engineered to express T6SS-5 in vitro show that the VgrG5 C-terminal domain is dispensable for T6SS-mediated secretion of Hcp5, demonstrating that the ability of VgrG5 to mediate membrane fusion can be uncoupled from its essential role in type VI secretion. We propose a model in which a unique fusogenic activity at the C terminus of VgrG5 facilitates intercellular spread by B. pseudomallei and related species following injection across the plasma membranes of infected cells.

  3. 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.

  4. YscP and YscU switch the substrate specificity of the Yersinia type III secretion system by regulating export of the inner rod protein YscI.

    PubMed

    Wood, Sarah E; Jin, Jin; Lloyd, Scott A

    2008-06-01

    Pathogenic yersiniae utilize a type III secretion system to inject antihost factors, called Yops, directly into the cytosol of eukaryotic cells. The Yops are injected via a needle-like structure, comprising the YscF protein, on the bacterial surface. While the needle is being assembled, Yops cannot be secreted. YscP and YscU switch the substrate specificity of the secretion system to enable Yop export once the needle attains its proper length. Here, we demonstrate that the inner rod protein YscI plays a critical role in substrate specificity switching. We show that YscI is secreted by the type III secretion system and that YscI secretion by a yscP mutant is abnormally elevated. Furthermore, we show that mutations in the cytoplasmic domain of YscU reduce YscI secretion by the yscP null strain. We also demonstrate that mutants expressing one of three forms of YscI (those with mutations Q84A, L87A, and L96A) secrete substantial amounts of Yops yet exhibit severe defects in needle formation. In the absence of YscP, mutants with the same changes in YscI assemble needles but are unable to secrete Yops. Together, these results suggest that the formation of the inner rod, not the needle, is critical for substrate specificity switching and that YscP and YscU exert their effects on substrate export by controlling the secretion of YscI.

  5. Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation.

    PubMed

    Bottai, Daria; Di Luca, Mariagrazia; Majlessi, Laleh; Frigui, Wafa; Simeone, Roxane; Sayes, Fadel; Bitter, Wilbert; Brennan, Michael J; Leclerc, Claude; Batoni, Giovanna; Campa, Mario; Brosch, Roland; Esin, Semih

    2012-03-01

    The chromosome of Mycobacterium tuberculosis encodes five type VII secretion systems (ESX-1-ESX-5). While the role of the ESX-1 and ESX-3 systems in M. tuberculosis has been elucidated, predictions for the function of the ESX-5 system came from data obtained in Mycobacterium marinum, where it transports PPE and PE_PGRS proteins and modulates innate immune responses. To define the role of the ESX-5 system in M. tuberculosis, in this study, we have constructed five M. tuberculosis H37Rv ESX-5 knockout/deletion mutants, inactivating eccA(5), eccD(5), rv1794 and esxM genes or the ppe25-pe19 region. Whereas the Mtbrv1794ko displayed no obvious phenotype, the other four mutants showed defects in secretion of the ESX-5-encoded EsxN and PPE41, a representative member of the large PPE protein family. Strikingly, the MtbeccD(5) ko mutant also showed enhanced sensitivity to detergents and hydrophilic antibiotics. When the virulence of the five mutants was evaluated, the MtbeccD(5) ko and MtbΔppe25-pe19 mutants were found attenuated both in macrophages and in the severe combined immune-deficient mouse infection model. Altogether these findings indicate an essential role of ESX-5 for transport of PPE proteins, cell wall integrity and full virulence of M. tuberculosis, thereby opening interesting new perspectives for the study of this human pathogen.

  6. Erwinia carotovora DsbA mutants: evidence for a periplasmic-stress signal transduction system affecting transcription of genes encoding secreted proteins.

    PubMed

    Vincent-Sealy, L V; Thomas, J D; Commander, P; Salmond, G P

    1999-08-01

    The dsbA genes, which encode major periplasmic disulfide-bond-forming proteins, were isolated from Erwinia carotovora subsp. carotovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca), and the dsbC gene, encoding another periplasmic disulfide oxidoreductase was isolated from Ecc. All three genes were sequenced and mutants deficient in these genes were created by marker exchange mutagenesis. The Ecc mutants were severely affected in activity and secretion of pectate lyase, probably due to the absence of functional PelC, which is predicted to require disulfide bond formation to achieve its correct conformation prior to secretion across the outer membrane. Similarly, endopolygalacturonase, also predicted to possess disulfide bonds, displayed reduced activity. The major Ecc cellulase (CelV) does not contain cysteine residues and was still secreted in dsbA-deficient strains. This observation demonstrated unequivocally that the localization and activity of the individual components of the Out apparatus are independent of disulfide bond formation. Surprisingly, cellulase activity was shown to be increased approximately two- to threefold in the DsbA mutant. This phenomenon resulted from transcriptional up-regulation of celV gene expression. In contrast, transcription of both pelC and peh were down-regulated in dsbA-deficient strains when compared to the wild-type. Protease (Prt) activity and secretion were unaffected in the Ecc dsbA mutant. Prt activity was considerably reduced in the double dsbA dsbC mutant. However Prt was secreted normally in this strain. The Eca dsbA mutant was found to be non-motile, suggesting that disulfide bond formation is essential for motility in this strain. All of the dsb mutants showed reduced tissue maceration in planta. These results suggest that a feedback regulation system operates in Ecc. In this system, defects in periplasmic disulfide bond formation act as a signal which is relayed to the transcription machinery regulating gene

  7. Systematic site-directed mutagenesis of the Helicobacter pylori CagL protein of the Cag type IV secretion system identifies novel functional domains

    PubMed Central

    Bönig, Tobias; Olbermann, Patrick; Bats, Simon H.; Fischer, Wolfgang; Josenhans, Christine

    2016-01-01

    The Cag Type IV secretion system, which contributes to inflammation and cancerogenesis during chronic infection, is one of the major virulence factors of the bacterial gastric pathogen Helicobacter pylori. We have generated and characterized a series of non-marked site-directed chromosomal mutants in H. pylori to define domains of unknown function of the essential tip protein CagL of the Cag secretion system. Characterizing the CagL mutants, we determined that their function to activate cells and transport the effector CagA was reduced to different extents. We identified three novel regions of the CagL protein, involved in its structural integrity, its possible interaction with the CagPAI T4SS pilus protein CagI, and in its binding to integrins and other host cell ligands. In particular two novel variable CagL motifs were involved in integrin binding, TSPSA, and TASLI, which is located opposite of its integrin binding motif RGD. We thereby defined functionally important subdomains within the CagL structure, which can be used to clarify CagL contributions in the context of other CagPAI proteins or for inhibition of the CagT4SS. This structure-function correlation of CagL domains can also be instructive for the functional characterization of other potential VirB5 orthologs whose structure is not yet known. PMID:27922023

  8. Unconventional protein secretion (UPS) pathways in plants.

    PubMed

    Ding, Yu; Robinson, David G; Jiang, Liwen

    2014-08-01

    As in yeast and mammalian cells, novel unconventional protein secretion (UPS) or unconventional membrane trafficking pathways are now known to operate in plants. UPS in plants is generally associated with stress conditions such as pathogen attack, but little is known about its underlying mechanism and function. Here, we present an update on the current knowledge of UPS in the plants in terms of its transport pathways, possible functions and its relationship to autophagy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Sequence-Based Prediction of Type III Secreted Proteins

    PubMed Central

    Arnold, Roland; Brandmaier, Stefan; Kleine, Frederick; Tischler, Patrick; Heinz, Eva; Behrens, Sebastian; Niinikoski, Antti; Mewes, Hans-Werner; Horn, Matthias; Rattei, Thomas

    2009-01-01

    The type III secretion system (TTSS) is a key mechanism for host cell interaction used by a variety of bacterial pathogens and symbionts of plants and animals including humans. The TTSS represents a molecular syringe with which the bacteria deliver effector proteins directly into the host cell cytosol. Despite the importance of the TTSS for bacterial pathogenesis, recognition and targeting of type III secreted proteins has up until now been poorly understood. Several hypotheses are discussed, including an mRNA-based signal, a chaperon-mediated process, or an N-terminal signal peptide. In this study, we systematically analyzed the amino acid composition and secondary structure of N-termini of 100 experimentally verified effector proteins. Based on this, we developed a machine-learning approach for the prediction of TTSS effector proteins, taking into account N-terminal sequence features such as frequencies of amino acids, short peptides, or residues with certain physico-chemical properties. The resulting computational model revealed a strong type III secretion signal in the N-terminus that can be used to detect effectors with sensitivity of ∼71% and selectivity of ∼85%. This signal seems to be taxonomically universal and conserved among animal pathogens and plant symbionts, since we could successfully detect effector proteins if the respective group was excluded from training. The application of our prediction approach to 739 complete bacterial and archaeal genome sequences resulted in the identification of between 0% and 12% putative TTSS effector proteins. Comparison of effector proteins with orthologs that are not secreted by the TTSS showed no clear pattern of signal acquisition by fusion, suggesting convergent evolutionary processes shaping the type III secretion signal. The newly developed program EffectiveT3 (http://www.chlamydiaedb.org) is the first universal in silico prediction program for the identification of novel TTSS effectors. Our findings will

  10. Eukaryotic pathways targeted by the type III secretion system effector protein, BipC, involved in the intracellular lifecycle of Burkholderia pseudomallei

    PubMed Central

    Kang, Wen-Tyng; Vellasamy, Kumutha Malar; Vadivelu, Jamuna

    2016-01-01

    Burkholderia pseudomallei, the etiological agent for melioidosis, is known to secrete a type III secretion system (TTSS) protein into the host’s internal milieu. One of the TTSS effector protein, BipC, has been shown to play an important role in the B. pseudomallei pathogenesis. To identify the host response profile that was directly or indirectly regulated by this protein, genome-wide transcriptome approach was used to examine the gene expression profiles of infected mice. The transcriptome analysis of the liver and spleen revealed that a total of approximately 1,000 genes were transcriptionally affected by BipC. Genes involved in bacterial invasion, regulation of actin cytoskeleton, and MAPK signalling pathway were over-expressed and may be specifically regulated by BipC in vivo. These results suggest that BipC mainly targets pathways related to the cellular processes which could modulate the cellular trafficking processes. The host transcriptional response exhibited remarkable differences with and without the presence of the BipC protein. Overall, the detailed picture of this study provides new insights that BipC may have evolved to efficiently manipulate host-cell pathways which is crucial in the intracellular lifecycle of B. pseudomallei. PMID:27634329

  11. Yersinia enterocolitica type III secretion: evidence for the ability to transport proteins that are folded prior to secretion.

    PubMed

    Wilharm, Gottfried; Lehmann, Verena; Neumayer, Wibke; Trcek, Janja; Heesemann, Jürgen

    2004-07-12

    Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a type three secretion system (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2-3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of specific Yop chaperones (called Sycs). In this study we have fused YopE secretion signals of various length to the mouse dihydrofolate reductase (DHFR) in order to analyse the DHFR folding state prior to secretion. We could demonstrate that secretion-deficient as well as secretion-competent YopE-DHFR fusions complexed to SycE can be efficiently purified from Yersinia cytosol by affinity chromatography using methotrexate-agarose. This implies the folding of the DHFR fusion moiety despite SycE binding and contradicts the previously presented model of folding inhibition by chaperone binding. Secretion-deficient YopE-DHFR fusions caused severe jamming of the TTSS. This observation contradicts the co-translational secretion model. We present evidence that the Yersinia TTSS is familiar with the processing of transport substrates which are folded prior to secretion. We therefore predict that an unfoldase is involved in type III secretion.

  12. 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.

  13. 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

  14. Symbiotic implications of type III protein secretion machinery in Rhizobium.

    PubMed

    Viprey, V; Del Greco, A; Golinowski, W; Broughton, W J; Perret, X

    1998-06-01

    The symbiotic plasmid of Rhizobium sp. NGR234 carries a cluster of genes that encodes components of a bacterial type III secretion system (TTSS). In both animal and plant pathogens, the TTSS is an essential component of pathogenicity. Here, we show that secretion of at least two proteins (y4xL and NolX) is controlled by the TTSS of NGR234 and occurs after the induction with flavonoids. Polar mutations in two TTSS genes, rhcN and the nod-box controlled regulator of transcription y4xl, block the secretion of both proteins and strongly affect the ability of NGR234 to nodulate a variety of tropical legumes including Pachyrhizus tuberosus and Tephrosia vogelii.

  15. Secreted Protein Acidic and Rich in Cysteine in Ocular Tissue

    PubMed Central

    Scavelli, Kurt; Chatterjee, Ayan

    2015-01-01

    Abstract Secreted protein acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is the prototypical matricellular protein. Matricellular proteins are nonstructural secreted proteins that provide an integration between cells and their surrounding extracellular matrix (ECM). Regulation of the ECM is important in maintaining the physiologic function of tissues. Elevated levels of SPARC have been identified in a variety of diseases involving pathologic tissue remodeling, such as hepatic fibrosis, systemic sclerosis, and certain carcinomas. Within the eye, SPARC has been identified in the trabecular meshwork, lens, and retina. Studies have begun to show the role of SPARC in these tissues and its possible role, specifically in primary open-angle glaucoma, cataracts, and proliferative vitreoretinopathy. SPARC may, therefore, be a therapeutic target in the treatment of certain ocular diseases. Further investigation into the mechanism of action of SPARC will be necessary in the development of SPARC-targeted therapy. PMID:26167673

  16. Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag.

    PubMed

    Garcia, Julie Torruellas; Ferracci, Franco; Jackson, Michael W; Joseph, Sabrina S; Pattis, Isabelle; Plano, Lisa R W; Fischer, Wolfgang; Plano, Gregory V

    2006-10-01

    Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3beta kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3beta antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.

  17. The N-terminus of IpaB provides a potential anchor to the Shigella type III secretion system tip complex protein IpaD

    PubMed Central

    Dickenson, Nicholas E.; Arizmendi, Olivia; Patil, Mrinalini K.; Toth, Ronald T.; Middaugh, C. Russell; Picking, William D.; Picking, Wendy L.

    2014-01-01

    The type III secretion system (T3SS) is an essential virulence factor for Shigella flexneri, providing a conduit through which host-altering effectors are injected directly into a host cell to promote uptake. The type III secretion apparatus (T3SA) is comprised of a basal body, external needle, and regulatory tip complex. The nascent needle is a polymer of MxiH capped by a pentamer of invasion plasmid antigen D (IpaD). Exposure to bile salts (e.g. deoxycholate) causes a conformational change in IpaD and promotes recruitment of IpaB to the needle tip. It has been proposed that IpaB senses contact with host cell membranes, recruiting IpaC and inducing full secretion of T3SS effectors. While the steps of T3SA maturation and their external triggers have been identified, details of specific protein interactions and mechanisms have remained difficult to study due to the hydrophobic nature of the IpaB and IpaC translocator proteins. Here we explored the ability for a series of soluble N-terminal IpaB peptides to interact with IpaD. We found that DOC is required for the interaction and that a region of IpaB between residues 11–27 is required for maximum binding, which was confirmed in vivo. Furthermore, intramolecular FRET measurements indicated that movement of the IpaD distal domain away from the protein core accompanied the binding of IpaB11-226. Together these new findings provide important new insight into the interactions and potential mechanisms that define the maturation of the Shigella T3SA needle tip complex and provide a foundation for further studies probing T3SS activation. PMID:24236510

  18. Marker for type VI secretion system effectors

    PubMed Central

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

    2014-01-01

    Bacteria use diverse mechanisms to kill, manipulate, and compete with other cells. The recently discovered type VI secretion system (T6SS) is widespread in bacterial pathogens and used to deliver virulence effector proteins into target cells. Using comparative proteomics, we identified two previously unidentified T6SS effectors that contained a conserved motif. Bioinformatic analyses revealed that this N-terminal motif, named MIX (marker for type six effectors), is found in numerous polymorphic bacterial proteins that are primarily located in the T6SS genome neighborhood. We demonstrate that several MIX-containing proteins are T6SS effectors and that they are not required for T6SS activity. Thus, we propose that MIX-containing proteins are T6SS effectors. Our findings allow for the identification of numerous uncharacterized T6SS effectors that will undoubtedly lead to the discovery of new biological mechanisms. PMID:24927539

  19. 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.

  20. Comparative genomic analysis of evolutionarily conserved but functionally uncharacterized membrane proteins in archaea: Prediction of novel components of secretion, membrane remodeling and glycosylation systems.

    PubMed

    Makarova, Kira S; Galperin, Michael Y; Koonin, Eugene V

    2015-11-01

    A systematic comparative genomic analysis of all archaeal membrane proteins that have been projected to the last archaeal common ancestor gene set led to the identification of several novel components of predicted secretion, membrane remodeling, and protein glycosylation systems. Among other findings, most crenarchaea have been shown to encode highly diverged orthologs of the membrane insertase YidC, which is nearly universal in bacteria, eukaryotes, and euryarchaea. We also identified a vast family of archaeal proteins, including the C-terminal domain of N-glycosylation protein AglD, as membrane flippases homologous to the flippase domain of bacterial multipeptide resistance factor MprF, a bifunctional lysylphosphatidylglycerol synthase and flippase. Additionally, several proteins were predicted to function as membrane transporters. The results of this work, combined with our previous analyses, reveal an unexpected diversity of putative archaeal membrane-associated functional systems that remain to be functionally characterized. A more general conclusion from this work is that the currently available collection of archaeal (and bacterial) genomes could be sufficient to identify (almost) all widespread functional modules and develop experimentally testable predictions of their functions.

  1. SecretP: a new method for predicting mammalian secreted proteins.

    PubMed

    Yu, Lezheng; Guo, Yanzhi; Zhang, Zheng; Li, Yizhou; Li, Menglong; Li, Gongbing; Xiong, Wenjia; Zeng, Yuhong

    2010-04-01

    In contrast to a large number of classically secreted proteins (CSPs) and non-secreted proteins (NSPs), only a few proteins have been experimentally proved to enter non-classical secretory pathways. So it is difficult to identify non-classically secreted proteins (NCSPs), and no methods are available for distinguishing the three types of proteins simultaneously. In order to solve this problem, a data mining has been taken firstly, and mammalian proteins exported via ER-Golgi-independent pathways are collected through extensive literature searches. In this paper, a support vector machine (SVM)-based ternary classifier named SecretP is proposed to predict mammalian secreted proteins by using pseudo-amino acid composition (PseAA) and five additional features. When distinguishing the three types of proteins, SecretP yielded an accuracy of 88.79%. Evaluating the performance of our method by an independent test set of 92 human proteins, 76 of them are correctly predicted as NCSPs. When performed on another public independent data set, the prediction result of SecretP is comparable to those of other existing computational methods. Therefore, SecretP can be a useful supplementary tool for future secretome studies. The web server SecretP and all supplementary tables listed in this paper are freely available at http://cic.scu.edu.cn/bioinformatics/secretp/index.htm. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  2. 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

  3. Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production

    PubMed Central

    Le Loir, Yves; Azevedo, Vasco; Oliveira, Sergio C; Freitas, Daniela A; Miyoshi, Anderson; Bermúdez-Humarán, Luis G; Nouaille, Sébastien; Ribeiro, Luciana A; Leclercq, Sophie; Gabriel, Jane E; Guimaraes, Valeria D; Oliveira, Maricê N; Charlier, Cathy; Gautier, Michel; Langella, Philippe

    2005-01-01

    Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species. PMID:15631634

  4. Regulation of Protein Secretion Through Controlled Aggregation in the Endoplasmic Reticulum

    NASA Astrophysics Data System (ADS)

    Rivera, Victor M.; Wang, Xiurong; Wardwell, Scott; Courage, Nancy L.; Volchuk, Allen; Keenan, Terence; Holt, Dennis A.; Gilman, Michael; Orci, Lelio; Cerasoli, Frank; Rothman, James E.; Clackson, Tim

    2000-02-01

    A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

  5. BcsKC is an essential protein for the type VI secretion system activity in Burkholderia cenocepacia that forms an outer membrane complex with BcsLB.

    PubMed

    Aubert, Daniel; MacDonald, Douglas K; Valvano, Miguel A

    2010-11-12

    The type VI secretion system (T6SS) contributes to the virulence of Burkholderia cenocepacia, an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. BcsK(C) is a highly conserved protein among the T6SSs in Gram-negative bacteria. Here, we show that BcsK(C) is required for Hcp secretion and cytoskeletal redistribution in macrophages upon bacterial infection. These two phenotypes are associated with a functional T6SS in B. cenocepacia. Experiments employing a bacterial two-hybrid system and pulldown assays demonstrated that BcsK(C) interacts with BcsL(B), another conserved T6SS component. Internal deletions within BcsK(C) revealed that its N-terminal domain is necessary and sufficient for interaction with BcsL(B). Fractionation experiments showed that BcsK(C) can be in the cytosol or tightly associated with the outer membrane and that BcsK(C) and BcsL(B) form a high molecular weight complex anchored to the outer membrane that requires BcsF(H) (a ClpV homolog) to be assembled. Together, our data show that BcsK(C)/BcsL(B) interaction is essential for the T6SS activity in B. cenocepacia.

  6. Cag-delta (Cag3) protein from the Helicobacter pylori 26695 cag type IV secretion system forms ring-like supramolecular assemblies.

    PubMed

    Smart, Jonathan; Fouillen, Aurélien; Casu, Bastien; Nanci, Antonio; Baron, Christian

    2017-01-01

    Helicobacter pylori is an important cause of gastric pathologies and persistent infection can lead to stomach cancer. Virulent H. pylori strains encode a type IV secretion system responsible for translocation of the oncogenic CagA protein into cells of the gastric mucosa. Gene HP0522 encodes the essential component Cagδ (Cag3), and we show by gel filtration and cross-linking that purified Cagδ forms high molecular mass complexes. In contrast, its interaction partner CagT is mostly monomeric, but co-fractionates after gel filtration. Analysis by transmission electron microscopy revealed that purified Cagδ complexes can self-assemble ring-like structures. Cagδ-overexpressing Escherichia coli exhibits membrane-associated circular profiles in regions of the cell envelope with intense immunogold labelling with a Cagδ-specific antiserum. Our results suggest that Cagδ has the capacity to form macromolecular structures contributing to the assembly of the type IV secretion system.

  7. High level heterologous protein production in Lactococcus and Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals.

    PubMed

    Savijoki, K; Kahala, M; Palva, A

    1997-02-28

    A secretion cassette, based on the expression and secretion signals of a S-layer protein (SlpA) from Lactobacillus brevis, was constructed. E. coli beta-lactamase (Bla) was used as the reporter protein to determine the functionality of the S-layer signals for heterologous expression and secretion in Lactococcus lactis, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus casei using a low-copy-number plasmid derived from pGK12. In all hosts tested, the bla gene was expressed under the slpA signals and all Bla activity was secreted to the culture medium. The Lb. brevis S-layer promoters were very efficiently recognized in L. lactis, Lb. brevis and Lb. plantarum, whereas in Lb. gasseri the slpA promoter region appeared to be recognized at a lower level and in Lb. casei the level of transcripts was below the detection limit. The production of Bla was mainly restricted to the exponential phase of growth. The highest yield of Bla was obtained with L. lactis and Lb. brevis. Without pH control, substantial degradation of Bla occurred during prolonged cultivations with all lactic acid bacteria (LAB) tested. When growing L. lactis and Lb. brevis under pH control, the Bla activity could be stabilized also at the stationary phase. L. lactis produced up to 80 mg/l of Bla which to our knowledge represents the highest amount of a heterologous protein secreted by LAB so far. The short production phase implied a very high rate of secretion with a calculated value of 5 x 10(5) Bla molecules/cell per h. Such a high rate was also observed with Lb. plantarum, whereas in Lb. brevis the competition between the wild type slpA gene and the secretion construct probably lowered the rate of Bla production. The results obtained indicate wide applicability of the Lb. brevis slpA signals for efficient protein production and secretion in LAB.

  8. Pachytene spermatocytes regulate the secretion of Sertoli cell protein(s) which stimulate Leydig cell steroidogenesis.

    PubMed

    Onoda, M; Djakiew, D; Papadopoulos, V

    1991-05-01

    The influence of germ cells (pachytene spermatocytes and round spermatids) on the secretion by Sertoli cells of the proteinaceous factor(s) which stimulates Leydig cell steroid biosynthesis was investigated. Sertoli cells from immature rats were cultured on plastic dishes or on Millipore filters impregnated with reconstituted basement membrane in bicameral chambers. Immature rat Sertoli cell secreted proteins (rSCSP; MW greater than 10,000), from conventional cultures, stimulated 4- to 5-fold steroid biosynthesis in normal rat and MA-10 mouse tumor Leydig cells, respectively. MA-10 cells were then used as a bioassay system for most studies, although purified rat Leydig cells were used in some cases to further confirm results obtained with MA-10 cells. rSCSP collected from both the apical and basal compartment of the chambers were examined for their ability to stimulate Leydig cell steroidogenesis. The Leydig cell stimulatory activity from Sertoli cells was found to be secreted in a polarized manner, with 80% of the total bioactivity found in the basal rSCSP. Addition of pachytene spermatocyte proteins (PSP) in the apical compartment of the chambers inhibited, in a time- and concentration-dependent manner, the basally directed Sertoli cell secretion of the Leydig cell stimulatory protein(s) by 85%. Similar results were obtained when freshly isolated pachytene spermatocytes were directly added on top of Sertoli cell epithelial sheets in the apical compartment of the chambers. In contrast, round spermatid proteins (RSP) did not exhibit a comparable effect to that of PSP in regulating the Sertoli cell secretion of the Leydig cell stimulatory activity. These results demonstrate that the Sertoli cell secreted protein(s) which stimulates Leydig cell steroid biosynthesis is secreted in a basally polarized direction, and its secretion is specifically modulated by pachytene spermatocytes.

  9. Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

    Treesearch

    Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

    2003-01-01

    Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

  10. 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

  11. Unique substrates secreted by the type VI secretion system of Francisella tularensis during intramacrophage infection.

    PubMed

    Bröms, Jeanette E; Meyer, Lena; Sun, Kun; Lavander, Moa; Sjöstedt, Anders

    2012-01-01

    Gram-negative bacteria have evolved sophisticated secretion machineries specialized for the secretion of macromolecules important for their life cycles. The Type VI secretion system (T6SS) is the most widely spread bacterial secretion machinery and is encoded by large, variable gene clusters, often found to be essential for virulence. The latter is true for the atypical T6SS encoded by the Francisella pathogenicity island (FPI) of the highly pathogenic, intracellular bacterium Francisella tularensis. We here undertook a comprehensive analysis of the intramacrophage secretion of the 17 FPI proteins of the live vaccine strain, LVS, of F. tularensis. All were expressed as fusions to the TEM β-lactamase and cleavage of the fluorescent substrate CCF2-AM, a direct consequence of the delivery of the proteins into the macrophage cytosol, was followed over time. The FPI proteins IglE, IglC, VgrG, IglI, PdpE, PdpA, IglJ and IglF were all secreted, which was dependent on the core components DotU, VgrG, and IglC, as well as IglG. In contrast, the method was not directly applicable on F. novicida U112, since it showed very intense native β-lactamase secretion due to FTN_1072. Its role was proven by ectopic expression in trans in LVS. We did not observe secretion of any of the LVS substrates VgrG, IglJ, IglF or IglI, when tested in a FTN_1072 deficient strain of F. novicida, whereas IglE, IglC, PdpA and even more so PdpE were all secreted. This suggests that there may be fundamental differences in the T6S mechanism among the Francisella subspecies. The findings further corroborate the unusual nature of the T6SS of F. tularensis since almost all of the identified substrates are unique to the species.

  12. Unique Substrates Secreted by the Type VI Secretion System of Francisella tularensis during Intramacrophage Infection

    PubMed Central

    Bröms, Jeanette E.; Sjöstedt, Anders

    2012-01-01

    Gram-negative bacteria have evolved sophisticated secretion machineries specialized for the secretion of macromolecules important for their life cycles. The Type VI secretion system (T6SS) is the most widely spread bacterial secretion machinery and is encoded by large, variable gene clusters, often found to be essential for virulence. The latter is true for the atypical T6SS encoded by the Francisella pathogenicity island (FPI) of the highly pathogenic, intracellular bacterium Francisella tularensis. We here undertook a comprehensive analysis of the intramacrophage secretion of the 17 FPI proteins of the live vaccine strain, LVS, of F. tularensis. All were expressed as fusions to the TEM β-lactamase and cleavage of the fluorescent substrate CCF2-AM, a direct consequence of the delivery of the proteins into the macrophage cytosol, was followed over time. The FPI proteins IglE, IglC, VgrG, IglI, PdpE, PdpA, IglJ and IglF were all secreted, which was dependent on the core components DotU, VgrG, and IglC, as well as IglG. In contrast, the method was not directly applicable on F. novicida U112, since it showed very intense native β-lactamase secretion due to FTN_1072. Its role was proven by ectopic expression in trans in LVS. We did not observe secretion of any of the LVS substrates VgrG, IglJ, IglF or IglI, when tested in a FTN_1072 deficient strain of F. novicida, whereas IglE, IglC, PdpA and even more so PdpE were all secreted. This suggests that there may be fundamental differences in the T6S mechanism among the Francisella subspecies. The findings further corroborate the unusual nature of the T6SS of F. tularensis since almost all of the identified substrates are unique to the species. PMID:23185631

  13. 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

  14. Folding kinetics and thermodynamics of Pseudomonas syringae effector protein AvrPto provide insight into translocation via the type III secretion system.

    PubMed

    Dawson, Jennifer E; Nicholson, Linda K

    2008-07-01

    In order to infect their hosts, many Gram-negative bacteria translocate agents of infection, called effector proteins, through the type III secretion system (TTSS) into the host cytoplasm. This process is thought to require at least partial unfolding of these agents, raising the question of how an effector protein might unfold to enable its translocation and then refold once it reaches the host cytoplasm. AvrPto is a well-studied effector protein of Pseudomonas syringae pv tomato. The presence of a readily observed unfolded population of AvrPto in aqueous solution and the lack of a known secretion chaperone make it ideal for studying the kinetic and thermodynamic characteristics that facilitate translocation. Application of Nzz exchange spectroscopy revealed a global, two-state folding equilibrium with 16% unfolded population, a folding rate of 1.8 s(-1), and an unfolding rate of 0.33 s(-1) at pH 6.1. TrAvrPto stability increases with increasing pH, with only 2% unfolded population observed at pH 7.0. The R(1) relaxation of TrAvrPto, which is sensitive to both the global anisotropy of folded TrAvrPto and slow exchange between folded and unfolded conformations, provided independent verification of the global kinetic rate constants. Given the acidic apoplast in which the pathogen resides and the more basic host cytoplasm, these results offer an intriguing mechanism by which the pH dependence of stability and slow folding kinetics of AvrPto would allow efficient translocation of the unfolded form through the TTSS and refolding into its functional folded form once inside the host.

  15. DBSecSys: A Database of Burkholderia mallei Secretion Systems

    DTIC Science & Technology

    2014-07-16

    toxins and the lipases, as well as non-proteinaceous substrates, e.g., cyclic β- glucans and polysaccharides. 2* - Represents a Sec/Tat-dependent system...divided into three types: 1) the archetypal bacterial proteins exported into the periplasm via the Sec system; 2) trimeric proteins with a single beta ...barrel domain; and 3) pairs of proteins in which one partner carries the beta barrel domain and the other partner is the secreted protein. 6

  16. HrpG and HrpV proteins from the Type III secretion system of Erwinia amylovora form a stable heterodimer.

    PubMed

    Gazi, Anastasia D; Charova, Spyridoula; Aivaliotis, Michalis; Panopoulos, Nicholas J; Kokkinidis, Michael

    2015-01-01

    Bacterial type III secretion systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or directly into eukaryotic host cell cytoplasm. Erwinia amylovora, the main agent of rosaceous plants fireblight disease, employs an Hrp/Hrc1 T3SS to accomplish its pathogenesis. The regulatory network that controls the activation of this T3SS is largely unknown in E. amylovora. However, in Pseudomonas syringae pathovars, the HrpG/HrpV complex has been shown to directly regulate the activity of transcription factor HrpS and consequently the upregulation of the Hrp/Hrc1 T3SS related genes. In this work, we report the successful recombinant production and purification of a stable E. amylovora HrpG/HrpV complex, using pPROpET, a bicistronic expression vector. Furthermore, we present the first solution structure of this complex based on small-angle X-ray scattering data.

  17. 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.

  18. High-yield secretion of multiple client proteins in Aspergillus.

    PubMed

    Segato, Fernando; Damásio, André R L; Gonçalves, Thiago A; de Lucas, Rosymar C; Squina, Fabio M; Decker, Stephen R; Prade, Rolf 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. An Interaction between the Inner Rod Protein YscI and the Needle Protein YscF Is Required to Assemble the Needle Structure of the Yersinia Type Three Secretion System.

    PubMed

    Cao, Shi-Yang; Liu, Wan-Bin; Tan, Ya-Fang; Yang, Hui-Ying; Zhang, Ting-Ting; Wang, Tong; Wang, Xiao-Yi; Song, Ya-Jun; Yang, Rui-Fu; Du, Zong-Min

    2017-03-31

    The type III secretion system is a highly conserved virulence mechanism that is widely distributed in Gram-negative bacteria. It has a syringe-like structure composed of a multi-ring basal body that spans the bacterial envelope and a projecting needle that delivers virulence effectors into host cells. Here, we showed that the Yersinia inner rod protein YscI directly interacts with the needle protein YscF inside the bacterial cells and that this interaction depends on amino acid residues 83-102 in the carboxyl terminus of YscI. Alanine substitution of Trp-85 or Ser-86 abrogated the binding of YscI to YscF as well as needle assembly and the secretion of effectors (Yops) and the needle tip protein LcrV. However, yscI null mutants that were trans-complemented with YscI mutants that bind YscF still assembled the needle and secreted Yops, demonstrating that a direct interaction between YscF and YscI is critical for these processes. Consistently, YscI mutants that did not bind YscF resulted in greatly decreased HeLa cell cytotoxicity. Together, these results show that YscI participates in needle assembly by directly interacting with YscF. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Substrate recognition by the Yersinia type III protein secretion machinery.

    PubMed

    Ramamurthi, Kumaran S; Schneewind, Olaf

    2003-11-01

    Type III secretion is the designation given to those protein secretion pathways, primarily in pathogenic Gram-negative bacteria, whose secretion machinery components share an amino acid sequence homology to components of the flagellar basal body. In Yersinia spp., these secretion machineries inject virulence proteins called Yops into the cytosol of target macrophages in an effort to evade phagocytic killing. To date, a clear mechanism by which Yops are recognized by the type III secretion machinery has not been elucidated. Unlike most, if not all, previously characterized protein sorting pathways, the information that identifies Yops as substrates for secretion seems not to be wholly encoded within the Yop peptide sequence. In fact, it appears that at least some of this information is contained within yop mRNAs. This review summarizes recent observations that have been made in this unusual field and proposes models by which proteins may be initiated into this pathway.

  1. The Mosaic Type IV Secretion Systems

    PubMed Central

    Christie, Peter J.

    2016-01-01

    Escherichia coli and other Gram-negative and -positive bacteria employ type IV secretion systems (T4SSs) to translocate DNA and protein substrates, generally by contact-dependent mechanisms, to other cells. The T4SSs functionally encompass two major subfamilies, the conjugation systems and the effector translocators. The conjugation systems are responsible for interbacterial transfer of antibiotic resistance genes, virulence determinants, and genes encoding other traits of potential benefit to the bacterial host. The effector translocators are used by many Gram-negative pathogens for delivery of potentially hundreds of virulence proteins termed effectors to eukaryotic cells during infection. In E. coli and other species of Enterobacteriaceae, T4SSs identified to date function exclusively in conjugative DNA transfer. In these species, the plasmid-encoded systems can be classified as the P, F, and I types. The P-type systems are the simplest in terms of subunit composition and architecture, and members of this subfamily share features in common with the paradigmatic Agrobacterium tumefaciens VirB/VirD4 T4SS. This review will summarize our current knowledge of the E. coli systems and the A. tumefaciens P-type system, with emphasis on the structural diversity of the T4SSs. Ancestral P-, F-, and I-type systems were adapted throughout evolution to yield the extant effector translocators, and information about well-characterized effector translocators also is included to further illustrate the adaptive and mosaic nature of these highly versatile machines. PMID:27735785

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

    PubMed

    de Sain, Mara; Rep, Martijn

    2015-10-09

    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.

  3. 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

  4. 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

  5. The type II secretion system: biogenesis, molecular architecture and mechanism.

    PubMed

    Korotkov, Konstantin V; Sandkvist, Maria; Hol, Wim G J

    2012-04-02

    Many gram-negative bacteria use the sophisticated type II secretion system (T2SS) to translocate a wide range of proteins from the periplasm across the outer membrane. The inner-membrane platform of the T2SS is the nexus of the system and orchestrates the secretion process through its interactions with the periplasmic filamentous pseudopilus, the dodecameric outer-membrane complex and a cytoplasmic secretion ATPase. Here, recent structural and biochemical information is reviewed to describe our current knowledge of the biogenesis and architecture of the T2SS and its mechanism of action.

  6. 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

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

    PubMed Central

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

    2013-01-01

    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. Regulation by ToxR-Like Proteins Converges on vttRB Expression To Control Type 3 Secretion System-Dependent Caco2-BBE Cytotoxicity in Vibrio cholerae

    PubMed Central

    Miller, Kelly A.; Sofia, Madeline K.; Weaver, Jacob W. A.; Seward, Christopher H.

    2016-01-01

    ABSTRACT Genes carried on the type 3 secretion system (T3SS) pathogenicity island of Vibrio cholerae non-O1/non-O139 serogroup strain AM-19226 must be precisely regulated in order for bacteria to cause disease. Previously reported results showed that both T3SS function and the presence of bile are required to cause Caco2-BBE cell cytotoxicity during coculture with strain AM-19226. We therefore investigated additional parameters affecting in vitro cell death, including bacterial load and the role of three transmembrane transcriptional regulatory proteins, VttRA, VttRB, and ToxR. VttRA and VttRB are encoded on the horizontally acquired T3SS genomic island, whereas ToxR is encoded on the ancestral chromosome. While strains carrying deletions in any one of the three transcriptional regulatory genes are unable to cause eukaryotic cell death, the results of complementation studies point to a hierarchy of regulatory control that converges on vttRB expression. The data suggest both that ToxR and VttRA act upstream of VttRB and that modifying the level of either vttRA or vttRB expression can strongly influence T3SS gene expression. We therefore propose a model whereby T3SS activity and, hence, in vitro cytotoxicity are ultimately regulated by vttRB expression. IMPORTANCE In contrast to O1 and O139 serogroup V. cholerae strains that cause cholera using two main virulence factors (toxin-coregulated pilus [TCP] and cholera toxin [CT]), O39 serogroup strain AM-19226 uses a type 3 secretion system as its principal virulence mechanism. Although the regulatory network governing TCP and CT expression is well understood, the factors influencing T3SS-associated virulence are not. Using an in vitro mammalian cell model to investigate the role of three ToxR-like transmembrane transcriptional activators in causing T3SS-dependent cytotoxicity, we found that expression levels and a hierarchical organization were important for promoting T3SS gene expression. Furthermore, our results

  9. Diverse C-terminal sequences involved in Flavobacterium johnsoniae protein secretion.

    PubMed

    Kulkarni, Surashree S; Zhu, Yongtao; Brendel, Colton J; McBride, Mark J

    2017-04-10

    Flavobacterium johnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to family TIGR04183 (type-A CTDs). We functionally characterized diverse CTDs for secretion by the F. johnsoniae T9SS. Attachment of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign protein sfGFP that had a signal peptide at the amino terminus resulted in secretion across the outer membrane. In each case approximately 80 to 100 amino acids from the extreme carboxy-termini was needed for efficient secretion. Several type-A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting secretion of sfGFP by the F. johnsoniae T9SS. F. johnsoniae SprB requires the T9SS for secretion but lacks a type-A CTD. It has a conserved C-terminal domain belonging to family TIGR04131, which we refer to as a type-B CTD. The CTD of SprB was required for its secretion, but attachment of C-terminal regions of SprB of up to 1182 amino acids to sfGFP failed to result in secretion. Additional features outside of the C-terminal region of SprB may be required for its secretion.Importance Type IX protein secretion systems (T9SSs) are common in but limited to members of the phylum Bacteroidetes Most proteins that are secreted by T9SSs have conserved carboxy-terminal domains that belong to either protein domain family TIGR04183 (type-A CTDs) or TIGR04131 (type-B CTDs). Here we identify features of T9SS CTDs of F. johnsoniae that are required for protein secretion and demonstrate that type-A CTDs from distantly related members of the phylum function with the F. johnsoniae T9SS to secrete the foreign protein sfGFP. In contrast, type-B CTDs failed

  10. Interplay among Pseudomonas syringae HrpR, HrpS and HrpV proteins for regulation of the type III secretion system

    PubMed Central

    Jovanovic, Milija; Lawton, Edward; Schumacher, Jörg

    2014-01-01

    Pseudomonas syringae pv. tomato DC3000, a plant pathogenic gram-negative bacterium, employs the type III secretion system (T3SS) to cause disease in tomato and Arabidopsis and to induce the hypersensitive response in nonhost plants. The expression of T3SS is regulated by the HrpL extracytoplasmic sigma factor. Expression of HrpL is controlled by transcriptional activators HrpR and HrpS and negative regulator HrpV. In this study, we analysed the organization of HrpRS and HrpV regulatory proteins and interplay between them. We identified one key residue I26 in HrpS required for repression by HrpV. Substitution of I26 in HrpS abolishes its interaction with HrpV and impairs interactions between HrpS and HrpR and the self-association of HrpS. We show that HrpS self-associates and can associate simultaneously with HrpR and HrpV. We now propose that HrpS has a central role in the assembly of the regulatory HrpRSV complex. Deletion analysis of HrpR and HrpS proteins showed that C-terminal parts of HrpR and HrpS confer determinants indispensable for their self-assembly. PMID:24863420

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

    PubMed Central

    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 Ca2+ 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 Ca2+ concentration raising the question of the driving force of substrate secretion by T1SS in general. PMID:27616645

  12. 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.

  13. 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

  14. Inflammasome activation in response to the Yersinia type III secretion system requires hyperinjection of translocon proteins YopB and YopD.

    PubMed

    Zwack, Erin E; Snyder, Annelise G; Wynosky-Dolfi, Meghan A; Ruthel, Gordon; Philip, Naomi H; Marketon, Melanie M; Francis, Matthew S; Bliska, James B; Brodsky, Igor E

    2015-02-17

    Type III secretion systems (T3SS) translocate effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. However, recognition of T3SS activity by cytosolic pattern recognition receptors (PRRs) of the nucleotide-binding domain leucine rich repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Macrophages infected with Yersinia pseudotuberculosis strains lacking all known effectors or lacking the translocation regulator YopK induce rapid activation of both the canonical NLRP3 and noncanonical caspase-11 inflammasomes. While this inflammasome activation requires a functional T3SS, the precise signal that triggers inflammasome activation in response to Yersinia T3SS activity remains unclear. Effectorless strains of Yersinia as well as ΔyopK strains translocate elevated levels of T3SS substrates into infected cells. To dissect the contribution of pore formation and translocation to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions of the T3SS. Notably, YopD variants that abrogated translocation but not pore-forming activity failed to induce inflammasome activation. Furthermore, analysis of individual infected cells revealed that inflammasome activation at the single-cell level correlated with translocated levels of YopB and YopD themselves. Intriguingly, LcrH mutants that are fully competent for effector translocation but produce and translocate lower levels of YopB and YopD also fail to trigger inflammasome activation. Our findings therefore suggest that hypertranslocation of YopD and YopB is linked to inflammasome activation in response to the Yersinia T3SS. The innate immune response is critical to effective clearance of pathogens. Recognition of conserved virulence structures and activities by innate immune receptors such as NLRs

  15. High-throughput system for the presentation of secreted and surface-exposed proteins from Gram-positive bacteria in functional metagenomics studies.

    PubMed

    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.

  16. An intrinsic mechanism of secreted protein aging and turnover.

    PubMed

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

    2015-11-03

    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.

  17. 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

  18. The Hcp proteins fused with diverse extended-toxin domains represent a novel pattern of antibacterial effectors in type VI secretion systems.

    PubMed

    Ma, Jiale; Pan, Zihao; Huang, Jinhu; Sun, Min; Lu, Chengping; Yao, Huochun

    2017-01-06

    The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many bacterial species to target eukaryotic host cells or rival bacteria. Using a dynamic injection mechanism, diverse effectors can be delivered by T6SS directly into recipient cells. Here, we report a new family of T6SS effectors encoded by extended Hcps carrying diverse toxin domains. Bioinformatic analyses revealed that these Hcps with C-terminal extension toxins, designated as Hcp-ET, exist widely in the Enterobacteriaceae. To verify our findings, Hcp-ET1 was tested for its antibacterial effect, and showed effective inhibition of target cell growth via the predicted HNH-DNase activity by T6SS-dependent delivery. Further studies showed that Hcp-ET2 mediated interbacterial antagonism via a Tle1 phospholipase (encoded by DUF2235 domain) activity. Notably, comprehensive analyses of protein homology and genomic neighborhoods revealed that Hcp-ET3-4 is fused with 2 toxin domains (Pyocin S3 and Colicin-DNase) C-terminally, and its encoding gene is followed 3 duplications of the cognate immunity genes. However, some bacteria encode a separated hcp-et3 and an orphan et4 (et4O1) genes caused by a termination-codon mutation in the fusion region between Pyocin S3 and Colicin-DNase encoding fragments. Our results demonstrated that both of these toxins had antibacterial effects. Further, all duplications of the cognate immunity protein contributed to neutralize the DNase toxicity of Pyocin S3 and Colicin, which has not been reported previously. In conclusion, we propose that Hcp-ET proteins are polymorphic T6SS effectors, and thus present a novel encoding pattern of T6SS effectors.

  19. The archetype Pseudomonas aeruginosa proteins TssB and TagJ form a novel subcomplex in the bacterial type VI secretion system.

    PubMed

    Lossi, Nadine S; Manoli, Eleni; Simpson, Pete; Jones, Cerith; Hui, Kailyn; Dajani, Rana; Coulthurst, Sarah J; Freemont, Paul; Filloux, Alain

    2012-10-01

    In Pseudomonas aeruginosa three type VI secretion systems (T6SSs) coexist, called H1- to H3-T6SSs. Several T6SS components are proposed to be part of a macromolecular complex resembling the bacteriophage tail. The T6SS protein HsiE1 (TagJ) is unique to the H1-T6SS and absent from the H2- and H3-T6SSs. We demonstrate that HsiE1 interacts with a predicted N-terminal α-helix in HsiB1 (TssB) thus forming a novel subcomplex of the T6SS. HsiB1 is homologous to the Vibrio cholerae VipA component, which contributes to the formation of a bacteriophage tail sheath-like structure. We show that the interaction between HsiE1 and HsiB1 is specific and does not occur between HsiE1 and HsiB2. Proteins of the TssB family encoded in T6SS clusters lacking a gene encoding a TagJ-like component are often devoid of the predicted N-terminal helical region, which suggests co-evolution. We observe that a synthetic peptide corresponding to the N-terminal 20 amino acids of HsiB1 interacts with purified HsiE1 protein. This interaction is a common feature to other bacterial T6SSs that display a TagJ homologue as shown here with Serratia marcescens. We further show that hsiE1 is a non-essential gene for the T6SS and suggest that HsiE1 may modulate incorporation of HsiB1 into the T6SS.

  20. The type VI protein secretion system contributes to biofilm formation and seed-to-seedling transmission of Acidovorax citrulli on melon.

    PubMed

    Tian, Yanli; Zhao, Yuqiang; Wu, Xinrong; Liu, Fengquan; Hu, Baishi; Walcott, Ronald R

    2015-01-01

    The type VI protein secretion system (T6SS) is essential for the virulence of several Gram-negative bacteria. In this study, we identified a T6SS gene cluster in Acidovorax citrulli, a plant-pathogenic bacterium that causes bacterial fruit blotch (BFB) of cucurbits. One T6SS cluster, of approximately 25 kb in length and comprising 17 genes, was found in the A. citrulli AAC00-1 genome. Seventeen A. citrulli mutants were generated, each with a deletion of a single T6SS core gene. There were significant differences in BFB seed-to-seedling transmission between wild-type A. citrulli strain, xjl12, and ΔvasD, ΔimpK, ΔimpJ and ΔimpF mutants (71.71%, 9.83%, 8.41%, 7.15% and 5.99% BFB disease index, respectively). In addition, we observed that these four mutants were reduced in melon seed colonization and biofilm formation; however, they were not affected in virulence when infiltrated into melon seedling tissues. There were no significant differences in BFB seed-to-seedling transmission, melon tissue colonization and biofilm formation between xjl12 and the other 13 T6SS mutants. Overall, our results indicate that T6SS plays a role in seed-to-seedling transmission of BFB on melon.

  1. 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.

  2. 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.

  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. 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...

  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 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

  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

    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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  7. 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

  8. 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.

  9. 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

  10. Contact with cultured epithelial cells stimulates secretion of Salmonella typhimurium invasion protein InvJ.

    PubMed Central

    Zierler, M K; Galán, J E

    1995-01-01

    Contact of Salmonella typhimurium with cultured epithelial cells results in the assembly of surface appendages termed invasomes which are presumably required for the internalization of these organisms into host cells. The assembly of these structures requires the function of a dedicated protein secretion system encoded in the inv locus. We show in this report that contact of wild-type S. typhimurium with cultured Henle-407 cells stimulated the secretion of InvJ, a recently identified target of the inv-encoded type III protein secretion system. Stimulation of InvJ secretion also occurred upon bacterial contact with bovine calf serum-coated culture dishes but did not occur upon S. typhimurium contact with glutaraldehyde-fixed Henle-407 cells. The stimulation of InvJ secretion did not require de novo protein synthesis. Invasion-defective invC and invG mutants of S. typhimurium failed to secrete InvJ upon contact with live Henle-407 cells. In contrast, contact-dependent secretion of InvJ in S. typhimurium invE mutants occurred at levels equivalent to those of the wild type. These results indicate that the presence of Henle-407 cells and/or serum is capable of activating the type III secretion system encoded in the inv locus, further supporting the notion that Salmonella entry into cultured cells is the result of a biochemical cross-talk between the bacteria and the host cells. PMID:7558314

  11. Azide-resistant mutants in Acinetobacter calcoaceticus A2 are defective in protein secretion.

    PubMed

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

    1994-02-15

    Azide, an inhibitor of ATPase, and a specific inhibitor of protein export was used in order to select for protein secretion mutants in Acinetobacter calcoaceticus A2. Two such mutants were isolated that were azide-resistant and defective in the general protein transport system. The mutation also conferred additional phenotypic changes, including an inability to grow on minimal media or at 40 degrees C. The existence of protein secretion mutants with a selectable phenotype may be useful for the genetic study of protein export.

  12. 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.

  13. Identification of luminal and secreted proteins in bull epididymis.

    PubMed

    Belleannée, Clémence; Labas, Valérie; Teixeira-Gomes, Ana-Paula; Gatti, Jean Luc; Dacheux, Jean-Louis; Dacheux, Françoise

    2011-01-01

    The epididymis plays a major role in the acquisition of sperm fertility. In order to shed light on specific features of epididymal function in mammalian species, we characterized the luminal proteins (luminal proteome) and secreted proteins (secretome) in the bovine epididymis. We identified 172 different luminal proteins in 9 distinct epididymal regions. The concentration and secretory activity of luminal proteins were quantified throughout the epididymis. Among the most abundant secreted proteins, we found lipocalin 5, (LCN5), NADP(+)dependent prostaglandin dehydrogenase (PTGDS), Niemann-Pick disease type C2 protein (NPC2), glutathione peroxidase type 5 (GPX 5), clusterin (CLU), hexosaminidase B (HEXB) and galactosidase (GLB1), each of which is released in distinct epididymal regions. Gelsolin, (GSN) previously not described in mammalian epididymal fluid, appeared to be a major protein secreted exclusively in the distal region of the bovine epididymis, where fully mature spermatozoa are stored. Although the major epididymal proteins are conserved between mammalian species, this study highlights the specificity and mechanisms of protein processing of epididymal secretion in the bull. In addition, this study provides a major insight into the sequential changes occurring in the sperm environment while gaining fertilizing capacity and could provide new information for the future identification of potential fertility markers. Copyright © 2010 Elsevier B.V. All rights reserved.

  14. 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

  15. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  17. 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.

  18. 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

  19. 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.

  20. Two-Partner Secretion: Combining Efficiency and Simplicity in the Secretion of Large Proteins for Bacteria-Host and Bacteria-Bacteria Interactions.

    PubMed

    Guérin, Jeremy; Bigot, Sarah; Schneider, Robert; Buchanan, Susan K; Jacob-Dubuisson, Françoise

    2017-01-01

    Initially identified in pathogenic Gram-negative bacteria, the two-partner secretion (TPS) pathway, also known as Type Vb secretion, mediates the translocation across the outer membrane of large effector proteins involved in interactions between these pathogens and their hosts. More recently, distinct TPS systems have been shown to secrete toxic effector domains that participate in inter-bacterial competition or cooperation. The effects of these systems are based on kin vs. non-kin molecular recognition mediated by specific immunity proteins. With these new toxin-antitoxin systems, the range of TPS effector functions has thus been extended from cytolysis, adhesion, and iron acquisition, to genome maintenance, inter-bacterial killing and inter-bacterial signaling. Basically, a TPS system is made up of two proteins, the secreted TpsA effector protein and its TpsB partner transporter, with possible additional factors such as immunity proteins for protection against cognate toxic effectors. Structural studies have indicated that TpsA proteins mainly form elongated β helices that may be followed by specific functional domains. TpsB proteins belong to the Omp85 superfamily. Open questions remain on the mechanism of protein secretion in the absence of ATP or an electrochemical gradient across the outer membrane. The remarkable dynamics of the TpsB transporters and the progressive folding of their TpsA partners at the bacterial surface in the course of translocation are thought to be key elements driving the secretion process.

  1. Key proteins involved in insulin vesicle exocytosis and secretion

    PubMed Central

    Xiong, Qian-Yin; Yu, Cui; Zhang, Yao; Ling, Liefeng; Wang, Lizhuo; Gao, Jia-Lin

    2017-01-01

    In vivo insulin secretion is predominantly affected by blood glucose concentration, blood concentration of amino acids, gastrointestinal hormones and free nerve functional status, in addition to other factors. Insulin is one of the most important hormones in the body, and its secretion is precisely controlled by nutrients, neurotransmitters and hormones. The insulin exocytosis process is similar to the neurotransmitter release mechanism. There are various types of proteins and lipids that participate in the insulin secretory vesicle fusion process, such as soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, Ras-related proteins and vacuolar-type H+-ATPase (V-ATPase). Notably, the SNARE protein is the molecular basis of exocytotic activity. In the current review, the role of the vesicle membrane proteins (synaptobrevins, vesicle associated membrane proteins and target membrane proteins) and auxiliary proteins (Rab proteins and Munc-18 proteins) in vesicle fusion activity were summarized. A summary of these key proteins involved in insulin granule secretion will facilitate understanding of the pathogenesis of diabetes. PMID:28357064

  2. Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an E. coli cell-free system

    PubMed Central

    Welsh, John P.; Bonomo, Jeanne; Swartz, James R.

    2011-01-01

    The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in E. coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome binding portion of the E. coli protein Trigger Factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both verified for the fusion protein. When added to E. coli-based CFPS reactions, the TF-BiP fusion chaperone increased soluble yields of several protein fragments that are normally secreted through the ER and have poor solubility in typical CFPS reactions. For comparison, a fusion between TF and the native E. coli Hsp70, DnaK, was also constructed. This fusion was also biologically active and increased soluble yields of certain protein targets in CFPS. The TF-BiP fusion described in this study can be seen as a first step in reconstituting and better understanding ER folding pathways in the prokaryotic environment of E. coli CFPS. PMID:21351069

  3. Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an Escherichia coli cell-free system.

    PubMed

    Welsh, John P; Bonomo, Jeanne; Swartz, James R

    2011-08-01

    The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in Escherichia coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome-binding portion of the E. coli protein trigger factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both verified for the fusion protein. When added to E. coli-based CFPS reactions, the TF-BiP fusion chaperone increased soluble yields of several protein fragments that are normally secreted through the ER and have poor solubility in typical CFPS reactions. For comparison, a fusion between TF and the native E. coli Hsp70, DnaK, was also constructed. This fusion was also biologically active and increased soluble yields of certain protein targets in CFPS. The TF-BiP fusion described in this study can be seen as a first step in reconstituting and better understanding ER folding pathways in the prokaryotic environment of E. coli CFPS. Copyright © 2011 Wiley Periodicals, Inc.

  4. The wingless-related integration site-5a/secreted frizzled-related protein-5 system is dysregulated in human sepsis.

    PubMed

    Schulte, D M; Kragelund, D; Müller, N; Hagen, I; Elke, G; Titz, A; Schädler, D; Schumacher, J; Weiler, N; Bewig, B; Schreiber, S; Laudes, M

    2015-04-01

    Sepsis and type 2 diabetes exhibit insulin resistance as a common phenotype. In type 2 diabetes we and others have recently provided evidence that alterations of the proinflammatory wingless-related integration site (wnt)-5a/anti-inflammatory secreted frizzled-related protein (sFRP)-5 system are involved in the pathogenesis of insulin resistance. The aim of the present study was to investigate whether this novel cytokine system is dysregulated in human sepsis, which may indicate a potential mechanism linking inflammation to metabolism. In this single-centre prospective observational study, critically ill adult septic patients were examined and proinflammatory wnt5a and wnt5a inhibitor sFRP5 were measured in serum samples by enzyme-linked immunosorbent assay (ELISA) at admission to the intensive care unit (ICU) and 5 days later. Sixty sepsis patients were included, and 30 healthy individuals served as controls. Wnt5a levels were found to be increased significantly in septic patients compared to healthy controls (2·21 ± 0·33 versus 0·32 ± 0·03 ng/ml, P < 0·0001). In contrast, sFRP5 was not altered significantly in septic patients (19·72 ± 3·06 versus 17·48 ± 6·38 ng/ml, P = 0·07). On admission to the ICU, wnt5a levels exhibited a significant positive correlation with the leucocyte count (rs  = 0·3797, P = 0·004). Interestingly, in patients recovering from sepsis, wnt5a levels declined significantly within 5 days (2·17 ± 0·38-1·03 ± 0·28 ng/ml, P < 0·01). In contrast, if sepsis was worsening, wnt5a levels increased in the same time-period by trend (2·34 ± 0·59-3·25 ± 1·02 ng/ml, P > 0·05). sFRP5 levels did not change significantly throughout the study period. The wnt5a/sFRP5 system is altered in human sepsis and might therefore be of interest for future studies on molecular pathophysiology of this common human disease.

  5. 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.

  6. Type II secretion in Yersinia—a secretion system for pathogenicity and environmental fitness

    PubMed Central

    von Tils, Dominik; Blädel, Inga; Schmidt, M. Alexander; Heusipp, Gerhard

    2012-01-01

    In Yersinia species, type III secretion (T3S) is the most prominent and best studied secretion system and a hallmark for the infection process of pathogenic Yersinia species. Type II secretion (T2S), on the other hand, is less well-characterized, although all Yersinia species, pathogenic as well as non-pathogenic, possess one or even two T2S systems. The only Yersinia strain in which T2S has so far been studied is the human pathogenic strain Y. enterocolitica 1b. Mouse infection experiments showed that at least one of the two T2S systems of Y. enterocolitica 1b, termed Yts1, is involved in dissemination and colonization of deeper tissues like liver and spleen. Interestingly, in vitro studies revealed a complex regulation of the Yts1 system, which is mainly active at low temperatures and high Mg2+-levels. Furthermore, the functional characterization of the proteins secreted in vitro indicates a role of the Yts1 machinery in survival of the bacteria in an environmental habitat. In silico analyses identified Yts1 homologous systems in bacteria that are known as plant symbionts or plant pathogens. Thus, the recent studies point to a dual function of the Yts1 T2S systems, playing a role in virulence of humans and animals, as well as in the survival of the bacteria outside of the mammalian host. In contrast, the role of the second T2S system, Yts2, remains ill defined. Whereas the T3S system and its virulence-mediating role has been intensively studied, it might now be time to also focus on the T2S system and its role in the Yersinia lifestyle, especially considering that most of the Yersinia isolates are not found in infected humans but have been gathered from various environmental samples. PMID:23248779

  7. Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris

    PubMed Central

    2011-01-01

    Background The increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers. Results Based on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER) has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p), we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold) without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion. Conclusions The current study presents a systems biotechnology-based strategy for the engineering of the

  8. Assembly, structure, function and regulation of type III secretion systems.

    PubMed

    Deng, Wanyin; Marshall, Natalie C; Rowland, Jennifer L; McCoy, James M; Worrall, Liam J; Santos, Andrew S; Strynadka, Natalie C J; Finlay, B Brett

    2017-04-10

    Type III secretion systems (T3SSs) are protein transport nanomachines that are found in Gram-negative bacterial pathogens and symbionts. Resembling molecular syringes, T3SSs form channels that cross the bacterial envelope and the host cell membrane, which enable bacteria to inject numerous effector proteins into the host cell cytoplasm and establish trans-kingdom interactions with diverse hosts. Recent advances in cryo-electron microscopy and integrative imaging have provided unprecedented views of the architecture and structure of T3SSs. Furthermore, genetic and molecular analyses have elucidated the functions of many effectors and key regulators of T3SS assembly and secretion hierarchy, which is the sequential order by which the protein substrates are secreted. As essential virulence factors, T3SSs are attractive targets for vaccines and therapeutics. This Review summarizes our current knowledge of the structure and function of this important protein secretion machinery. A greater understanding of T3SSs should aid mechanism-based drug design and facilitate their manipulation for biotechnological applications.

  9. A Secretion-Amplification Role for Salmonella enterica Translocon Protein SipD.

    PubMed

    Glasgow, Anum Azam; Wong, Han Teng; Tullman-Ercek, Danielle

    2017-03-16

    The bacterial type III secretion system (T3SS) is an important target for enabling high-titer production of proteins of biotechnological interest as well as for synthetic biology applications that rely on protein delivery to host cells. The T3SS forms a membrane-embedded needle complex that is capped by the translocon proteins and extends into the extracellular space. The needle tip complex in Salmonella enterica consists of three translocon proteins: SipB, SipC, and SipD. It is known that knocking out sipD disrupts T3SS regulation to cause constitutive secretion of native proteins. However, we discovered that complementation of SipD in trans via exogenous addition to T3SS-expressing cultures further improves heterologous protein secretion titers, suggesting a previously unknown but important role for this protein. Building on this knowledge, we have engineered a hyper-secreting strain of S. enterica for a greater than 100-fold improvement in the production of a variety of biotechnologically valuable heterologous proteins that are challenging to produce, such as toxic antimicrobial peptides and proteolysis-prone biopolymer proteins. We determined that transcription by several T3SS promoters is upregulated with the addition of SipD, that the N-terminal domain of SipD is sufficient to observe the increased secretion phenotype, and that the effect is post-transcriptional and post-translational. These results lend support to the use of bacterial secretion as a powerful protein production strategy, and the hypothesis that translocon proteins contribute to type III secretion regulation.

  10. 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

  11. 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

  12. Excreted/Secreted Proteins from Trypanosome Procyclic Strains

    PubMed Central

    Atyame Nten, Celestine Michelle; Sommerer, Nicolas; Rofidal, Valerie; Hirtz, Christophe; Rossignol, Michel; Cuny, Gerard; Peltier, Jean-Benoit; Geiger, Anne

    2010-01-01

    Trypanosoma secretome was shown to be involved in parasite virulence and is suspected of interfering in parasite life-cycle steps such as establishment in the Glossina midgut, metacyclogenesis. Therefore, we attempted to identify the proteins secreted by procyclic strains of T. brucei gambiense and T. brucei brucei, responsible for human and animal trypanosomiasis, respectively. Using mass spectrometry, 427 and 483 nonredundant proteins were characterized in T. brucei brucei and T. brucei gambiense secretomes, respectively; 35% and 42% of the corresponding secretome proteins were specifically secreted by T. brucei brucei and T. brucei gambiense, respectively, while 279 proteins were common to both subspecies. The proteins were assigned to 12 functional classes. Special attention was paid to the most abundant proteases (14 families) because of their potential implication in the infection process and nutrient supply. The presence of proteins usually secreted via an exosome pathway suggests that this type of process is involved in trypanosome ESP secretion. The overall results provide leads for further research to develop novel tools for blocking trypanosome transmission. PMID:20011064

  13. Type III secretion systems shape up as they ship out.

    PubMed

    Marlovits, Thomas C; Stebbins, C Erec

    2010-02-01

    Virulence associated protein type III secretion systems (T3SSs) are intricately structured organic nanosyringes that achieve the translocation of bacterial proteins from the prokaryotic cytoplasm across three membranes into the host cytosol. The substrates for these systems number in the hundreds, with remarkably diverse biological activities, modulating host cell biology for the benefit of the pathogen. Although there has been tremendous progress on the structure and function of the T3SS substrates, there has been comparatively little progress on the much more highly conserved secretion apparatus itself. This review summarizes recent advances in the field of structural microbiology that have begun to address this shortcoming, finally bringing to bear the power of structural biology to this central virulence system of Gram-negative bacterial pathogens. Copyright 2009 Elsevier Ltd. All rights reserved.

  14. Secretion, delivery and function of oomycete effector proteins.

    PubMed

    Wawra, Stephan; Belmonte, Rodrigo; Löbach, Lars; Saraiva, Marcia; Willems, Ariane; van West, Pieter

    2012-12-01

    Oomycetes are responsible for multi-billion dollar damages in aquaculture, agriculture and forestry. One common strategy they share with most cellular disease agents is the secretion of effector proteins. Effectors are molecules that change host physiology by initiating and allowing an infection to develop. Oomycetes secrete both extracellular and intracellular effectors. Studying secretion, delivery and function of effectors will hopefully lead to alternative control measures, which is much needed as several chemicals to control plant and animal pathogenic oomycetes cannot be used anymore; due to resistance in the host, or because the control measures have been prohibited as a result of toxicity to the environment and/or consumers. Here the latest findings on oomycete effector secretion, delivery and function are discussed.

  15. 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

  16. Differences in mesentericin secretion systems from two Leuconostoc strains.

    PubMed

    Aucher, Willy; Simonet, Valérie; Fremaux, Christophe; Dalet, Karine; Simon, Laurence; Cenatiempo, Yves; Frère, Jacques; Berjeaud, Jean-Marc

    2004-03-12

    Leuconostoc mesenteroides Y105 and L. mesenteroides FR52 produce both mesentericin Y105 and B105, in equal amounts. The mesentericin operons of L. mesenteroides FR52 and Y105 which are involved in mesentericin Y105 and B105 production, were both sequenced and compared. Differences were limited to the two genes, mesD and mesE, which encode the dedicated transport system of mesentericin Y105. Analysis of mesentericin non-producing mutants and complementation experiments demonstrated that the major role of the membrane fusion protein, MesE, was in bacteriocin secretion for both strains. Moreover, the secretion machinery MesDE was demonstrated to be capable of transportation and maturation of the two pre-bacteriocins, mesentericin Y105 and B105. We also demonstrate that although MesDEs from strains Y105 and FR52 have significant sequence differences, both transporters were capable of assuring secretion of either bacteriocin.

  17. Crystal structure of YwpF from Staphylococcus aureus reveals its architecture comprised of a β-barrel core domain resembling type VI secretion system proteins and a two-helix pair.

    PubMed

    Lee, Sang Jae; Lee, Kyu-Yeon; Lee, Ki-Young; Kim, Dong-Gyun; Kim, Soon-Jong; Lee, Bong-Jin

    2015-04-01

    The ywpF gene (SAV2097) of the Staphylococcus aureus strain Mu50 encodes the YwpF protein, which may play a role in antibiotic resistance. Here, we report the first crystal structure of the YwpF superfamily from S. aureus at 2.5-Å resolution. The YwpF structure consists of two regions: an N-terminal core β-barrel domain that shows structural similarity to type VI secretion system (T6SS) proteins (e.g., Hcp1, Hcp3, and EvpC) and a C-terminal two-helix pair. Although the monomer structure of S. aureus YwpF resembles those of T6SS proteins, the dimer/tetramer model of S. aureus YwpF is distinct from the functionally important hexameric ring of T6SS proteins. We therefore suggest that the S. aureus YwpF may have a different function compared to T6SS proteins.

  18. TolC-dependent secretion of an ankyrin repeat-containing protein of Rickettsia typhi.

    PubMed

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

    2012-09-01

    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.

  19. Anaplasma phagocytophilum and Ehrlichia chaffeensis type IV secretion and Ank proteins

    PubMed Central

    RIKIHISA, YASUKO; LIN, MINGQUN

    2011-01-01

    Summary of recent advances The obligatory intracellular bacterial pathogens Anaplasma and Ehrlichia infect leukocytes by hijacking host-cell components and processes. The type IV secretion system is up-regulated during infection. Among type IV secretion candidate substrates, an ankyrin repeat protein of Anaplasma phagocytophilum, AnkA, is delivered into the host cytoplasm via a complex that includes VirD4. AnkA is highly tyrosine-phosphorylated and binds to the Abl interactor 1, SHP-1, and nuclear DNA fragments. Ehrlichia chaffeensis AnkA was recently reported to be translocated into host cell nucleus. The recent discovery of several ankyrin repeat proteins secreted via the type IV secretion system of different intracellular bacteria suggests that a common strategy evolved to subvert host-cell functions. PMID:20053580

  20. Secreted Proteins Defy the Expression Level-Evolutionary Rate Anticorrelation.

    PubMed

    Feyertag, Felix; Berninsone, Patricia M; Alvarez-Ponce, David

    2017-03-01

    The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E-R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein-protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E-R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E-R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E-R anticorrelation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops.

    PubMed Central

    Tardy, F; Homblé, F; Neyt, C; Wattiez, R; Cornelis, G R; Ruysschaert, J M; Cabiaux, V

    1999-01-01

    'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors. PMID:10581252

  2. Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops.

    PubMed

    Tardy, F; Homblé, F; Neyt, C; Wattiez, R; Cornelis, G R; Ruysschaert, J M; Cabiaux, V

    1999-12-01

    'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors.

  3. The Structure and Function of Type III Secretion Systems.

    PubMed

    Notti, Ryan Q; Stebbins, C Erec

    2016-02-01

    Type III secretion systems (T3SSs) afford Gram-negative bacteria an 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 posttranslational regulation of secretory function. Lastly, we close with a discussion of the future prospects for the interrogation of structure-function relationships in the T3SS.

  4. Cloning and characterization of three hypothetical secretion chaperone proteins from Xanthomonas axonopodis pv. citri.

    PubMed

    Tasic, Ljubica; Borin, Paula F L; Khater, Leti Cia; Ramos, Carlos H I

    2007-06-01

    Xanthomonas axonopodis pv. citri (Xac) causes citrus canker in plantations around the world and is of particular significance in Brazil where its incidence has risen exponentially over the past decade. Approximately one third of the predicted Xac open reading frames show no homology, or homology with very low score with that of known sequences. It is believed that Xac utilizes secretion systems to transfer virulence proteins into susceptible eukaryotic cells. This process is assisted by secretion chaperones that maintain virulence proteins partly or completely unfolded during translocation. We have cloned three of these hypothetical secretion chaperones: XAC0419 and XAC1346 from type III secretion system (TTSS) and XACb0033 from type IV secretion system (TFSS). All proteins were cloned in a pET23a vector (Novagen), expressed at 37 degrees C using a BL21(DE3)pLysS Escherichia coli strain and purified by ion exchange and gel-filtration chromatographic methods. Pure proteins were characterized using spectroscopic measurements: circular dichroism, and both static and lifetime emission fluorescence in the case of XACb0033. The analyzed proteins are stable at elevated temperatures (up to 65 degrees C) and exhibit alpha-helix content from approximately 30% (XACb003) to approximately 87% (XAC1346). XACb0033 exhibits lifetimes in the fluorescence experiments that indicate different neighborhoods for its tryptophan residues. These chaperones have the characteristics of TTSS and TFSS: all are small, with a high alpha-helix content, and without ATP-binding or ATP-hydrolyzing activity.

  5. Functional type 1 secretion system involved in Legionella pneumophila virulence.

    PubMed

    Fuche, Fabien; Vianney, Anne; Andrea, Claire; Doublet, Patricia; Gilbert, Christophe

    2015-02-01

    Legionella pneumophila is a Gram-negative pathogen found mainly in water, either in a free-living form or within infected protozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called legionellosis or Legionnaires' disease. The involvement of type II and IV secretion systems in the virulence of L. pneumophila is now well documented. Despite bioinformatic studies showing that a type I secretion system (T1SS) could be present in this pathogen, the functionality of this system based on the LssB, LssD, and TolC proteins has never been established. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle of L. pneumophila. Using deletion mutants and fusion proteins, we demonstrated that the repeats-in-toxin protein RtxA is secreted through an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these results underline the active participation of L. pneumophila, via its T1SS, in its internalization into host cells.

  6. Alternative protein secretion: The Mam1 ABC transporter supports secretion of M-factor linked GFP in fission yeast

    SciTech Connect

    Kjaerulff, Soren

    2005-12-30

    To examine whether the fission yeast Mam1 ABC transporter can be used for secretion of heterologous proteins, thereby bypassing the classical secretion pathway, we have analyzed chimeric forms of the M-factor precursor. It was demonstrated that GFP can be exported when fused to both the amino-terminal prosequence from mfm1 and a CaaX motif. This secretion was dependent on the Mam1 transporter and not the classical secretion pathway. The secretion efficiency of GFP, however, was relatively low and most of the reporter protein was trapped in the vacuolar membranes. Our findings suggest that the Mam1 ABC protein is a promiscuous peptide transporter that can accommodate globular proteins of a relatively large size. Furthermore, our results help in defining the sequences required for processing and secretion of natural M-factor.

  7. Immunogenic Domains and Secondary Structure of Escherichia coli Recombinant Secreted Protein Escherichia coli-Secreted Protein B

    PubMed Central

    Caetano, Bruna Alves; Rocha, Letícia Barboza; Carvalho, Eneas; Piazza, Roxane Maria Fontes; Luz, Daniela

    2017-01-01

    Several pathogenic bacteria are able to induce the attaching and effacing (A/E) lesion. The A/E lesion is caused by effector proteins, such as Escherichia coli-secreted protein B (EspB), responsible together with Escherichia coli-secreted protein D for forming a pore structure on the host cell, which allows the translocation of effector proteins. Different variants of this protein can be found in E. coli strains, and during natural infection or when this protein is injected, this leads to variant-specific production of antibodies, which may not be able to recognize other variants of this bacterial protein. Herein, we describe the production of a hybrid recombinant EspB toxin that comprises all known variants of this protein. This recombinant protein could be useful as an antigen for the production of antibodies with broad-range detection of EspB-bearing bacteria, or as an antigen that could be used in vaccine formulation to generate antibodies against different EspB variants, thereby increasing immunization potential. In addition, the recombinant protein allowed us to analyze its secondary structure, to propose the immunogenic regions of EspB variants, and also to characterize anti-EspB antibodies. Our results suggest that this hybrid protein or a protein composed of the conserved immunogenic regions could be used for a variety of clinical applications. PMID:28484467

  8. Immunogenic Domains and Secondary Structure of Escherichia coli Recombinant Secreted Protein Escherichia coli-Secreted Protein B.

    PubMed

    Caetano, Bruna Alves; Rocha, Letícia Barboza; Carvalho, Eneas; Piazza, Roxane Maria Fontes; Luz, Daniela

    2017-01-01

    Several pathogenic bacteria are able to induce the attaching and effacing (A/E) lesion. The A/E lesion is caused by effector proteins, such as Escherichia coli-secreted protein B (EspB), responsible together with Escherichia coli-secreted protein D for forming a pore structure on the host cell, which allows the translocation of effector proteins. Different variants of this protein can be found in E. coli strains, and during natural infection or when this protein is injected, this leads to variant-specific production of antibodies, which may not be able to recognize other variants of this bacterial protein. Herein, we describe the production of a hybrid recombinant EspB toxin that comprises all known variants of this protein. This recombinant protein could be useful as an antigen for the production of antibodies with broad-range detection of EspB-bearing bacteria, or as an antigen that could be used in vaccine formulation to generate antibodies against different EspB variants, thereby increasing immunization potential. In addition, the recombinant protein allowed us to analyze its secondary structure, to propose the immunogenic regions of EspB variants, and also to characterize anti-EspB antibodies. Our results suggest that this hybrid protein or a protein composed of the conserved immunogenic regions could be used for a variety of clinical applications.

  9. Flavobacterium johnsoniae Chitinase ChiA Is Required for Chitin Utilization and Is Secreted by the Type IX Secretion System

    PubMed Central

    Kharade, Sampada S.

    2014-01-01

    Flavobacterium johnsoniae, a member of phylum Bacteriodetes, is a gliding bacterium that digests insoluble chitin and many other polysaccharides. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding motility and for chitin utilization. Five potential chitinases were identified by genome analysis. Fjoh_4555 (ChiA), a 168.9-kDa protein with two glycoside hydrolase family 18 (GH18) domains, was targeted for analysis. Disruption of chiA by insertional mutagenesis resulted in cells that failed to digest chitin, and complementation with wild-type chiA on a plasmid restored chitin utilization. Antiserum raised against recombinant ChiA was used to detect the protein and to characterize its secretion by F. johnsoniae. ChiA was secreted in soluble form by wild-type cells but remained cell associated in strains carrying mutations in any of the T9SS genes, gldK, gldL, gldM, gldNO, sprA, sprE, and sprT. Western blot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses suggested that ChiA was proteolytically processed into two GH18 domain-containing proteins. Proteins secreted by T9SSs typically have conserved carboxy-terminal domains (CTDs) belonging to the TIGRFAM families TIGR04131 and TIGR04183. ChiA does not exhibit strong similarity to these sequences and instead has a novel CTD. Deletion of this CTD resulted in accumulation of ChiA inside cells. Fusion of the ChiA CTD to recombinant mCherry resulted in secretion of mCherry into the medium. The results indicate that ChiA is a soluble extracellular chitinase required for chitin utilization and that it relies on a novel CTD for secretion by the F. johnsoniae T9SS. PMID:24363341

  10. Flavobacterium johnsoniae chitinase ChiA is required for chitin utilization and is secreted by the type IX secretion system.

    PubMed

    Kharade, Sampada S; McBride, Mark J

    2014-03-01

    Flavobacterium johnsoniae, a member of phylum Bacteriodetes, is a gliding bacterium that digests insoluble chitin and many other polysaccharides. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding motility and for chitin utilization. Five potential chitinases were identified by genome analysis. Fjoh_4555 (ChiA), a 168.9-kDa protein with two glycoside hydrolase family 18 (GH18) domains, was targeted for analysis. Disruption of chiA by insertional mutagenesis resulted in cells that failed to digest chitin, and complementation with wild-type chiA on a plasmid restored chitin utilization. Antiserum raised against recombinant ChiA was used to detect the protein and to characterize its secretion by F. johnsoniae. ChiA was secreted in soluble form by wild-type cells but remained cell associated in strains carrying mutations in any of the T9SS genes, gldK, gldL, gldM, gldNO, sprA, sprE, and sprT. Western blot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses suggested that ChiA was proteolytically processed into two GH18 domain-containing proteins. Proteins secreted by T9SSs typically have conserved carboxy-terminal domains (CTDs) belonging to the TIGRFAM families TIGR04131 and TIGR04183. ChiA does not exhibit strong similarity to these sequences and instead has a novel CTD. Deletion of this CTD resulted in accumulation of ChiA inside cells. Fusion of the ChiA CTD to recombinant mCherry resulted in secretion of mCherry into the medium. The results indicate that ChiA is a soluble extracellular chitinase required for chitin utilization and that it relies on a novel CTD for secretion by the F. johnsoniae T9SS.

  11. Inhibition of ERK1/2 Pathway Suppresses Adiponectin Secretion via Accelerating Protein Degradation by Ubiquitin-Proteasome System: Relevance to Obesity-related Adiponectin Decline

    PubMed Central

    Gu, Dongfang; Wang, Zhigang; Dou, Xiaobing; Zhang, Ximei; Li, Songtao; Vu, Lyndsey; Yao, Tong; Song, Zhenyuan

    2013-01-01

    Objective Predominantly secreted by adipose tissue, adiponectin possesses insulin-sensitizing, anti-atherogenic, anti-inflammatory, and anti-angiogenic properties. Paradoxically, obesity is associated with declined plasma adiponectin levels; however, the underlying mechanisms remain elusive. In this study, we investigated the mechanistic involvement of MEK/ERK1/2 pathway in obesity-related adiponectin decrease. Materials/Methods C57 BL/6 mice exposed to a high-fat diet (HFD) were employed as animal obesity model. Both fully-differentiated 3T3-L1 and mouse primary adipocytes were used in the in vitro experiments. Results Obesity and plasma adiponectin decline induced by prolonged HFD exposure was associated with suppressed ERK1/2 activation in adipose tissue. In adipocytes, specific inhibition of MEK/ERK1/2 pathway decreased intracellular and secretory adiponectin levels, whereas adiponectin gene expression was increased, suggesting that MEK/ERK1/2 inhibition may promote adiponectin protein degradation. Cycloheximide (CHX)-chase assay revealed that MEK/ERK1/2 inhibition accelerated adiponectin protein degradation, which was prevented by MG132, a potent proteasome inhibitor. Immunoprecipitation assay showed that intracellular MEK/ERK1/2 activity was negatively associated with ubiqutinated adiponectin protein levels. Consistently, long-term HFD feeing in mice increased ubiquitinated adiponectin levels in the epididymal fat pads. Conclusions Adipose tissue MEK/ERK1/2 activity can differentially regulates adiponectin gene expression and protein abundance and its suppression in obesity may play a mechanistic role in obesity-related plasma adiponectin decline. PMID:23490586

  12. Expression and secretion of the heterodimeric protein interleukin-12 in plant cell suspension culture.

    PubMed

    Kwon, T H; Seo, J E; Kim, J; Lee, J H; Jang, Y S; Yang, M S

    2003-03-30

    It has been suggested that plant cell culture is the most suitable system for producing small-to-medium quantities of specialized, expensive, and high-purity proteins. Here, we report that a heterodimeric protein, human interleukin-12 (hIL-12), was expressed and secreted into culture medium in a biologically active form. A transgenic plant expressing hIL-12 was constructed by sexual crossing of plants that expressed each subunit of the protein. From a piece of transgenic plant, callus was induced and cell suspension culture was established. The biological activity and amount of hIL-12 secreted into culture medium were analyzed using bioassays and ELISA. Analysis of cellular localization demonstrated that the protein was secreted into the culture medium together with its intrinsic signal peptide. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 870-875, 2003.

  13. The Bordetella pertussis Type III Secretion System Tip Complex Protein Bsp22 Is Not a Protective Antigen and Fails To Elicit Serum Antibody Responses during Infection of Humans and Mice

    PubMed Central

    Villarino Romero, Rodrigo; Bibova, Ilona; Cerny, Ondrej; Vecerek, Branislav; Wald, Tomas; Benada, Oldrich; Zavadilova, Jana; Sebo, Peter

    2013-01-01

    The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice. PMID:23690400

  14. Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis.

    PubMed

    Stock, Janpeter; Sarkari, Parveen; Kreibich, Saskia; Brefort, Thomas; Feldbrügge, Michael; Schipper, Kerstin

    2012-10-15

    The demand on the biotechnological production of proteins for pharmaceutical, medical and industrial applications is steadily growing. For the production of challenging proteins, we aim to establish a novel expression platform in the well characterized eukaryotic microorganism Ustilago maydis. In filaments of this fungus, secretion of the endochitinase Cts1 depends on mRNA transport along microtubules, which is mediated by the key RNA-binding protein Rrm4. Here, we report two important findings: (i) Cts1 secretion occurs via a novel unconventional route and (ii) this secretory mechanism can be exploited for the export of active heterologous proteins. Initially, we used β-glucuronidase (Gus) as a reporter for unconventional secretion. This bacterial enzyme is inactivated by N-glycosylation during its passage through the conventional eukaryotic secretory pathway. By contrast, in our system Gus was exported in its active form by fusion to Cts1 confirming its secretion by an unconventional route. As a proof-of-principle for economically important biopharmaceuticals we expressed an active single-chain antibody. Importantly, the novel protein export pathway circumvents N-glycosylation which is advantageous in many applications, e.g., to avoid undesired immune reactions in humans. Thus, the unconventional Cts1 secretion machinery has a high potential for the production of biotechnologically relevant proteins.

  15. Comparative genomics of the type VI secretion systems of Pantoea and Erwinia species reveals the presence of putative effector islands that may be translocated by the VgrG and Hcp proteins

    PubMed Central

    2011-01-01

    Background The Type VI secretion apparatus is assembled by a conserved set of proteins encoded within a distinct locus. The putative effector proteins Hcp and VgrG are also encoded within these loci. We have identified numerous distinct Type VI secretion system (T6SS) loci in the genomes of several ecologically diverse Pantoea and Erwinia species and detected the presence of putative effector islands associated with the hcp and vgrG genes. Results Between two and four T6SS loci occur among the Pantoea and Erwinia species. While two of the loci (T6SS-1 and T6SS-2) are well conserved among the various strains, the third (T6SS-3) locus is not universally distributed. Additional orthologous loci are present in Pantoea sp. aB-valens and Erwinia billingiae Eb661. Comparative analysis of the T6SS-1 and T6SS-3 loci showed non-conserved islands associated with the vgrG and hcp, and vgrG genes, respectively. These regions had a G+C content far lower than the conserved portions of the loci. Many of the proteins encoded within the hcp and vgrG islands carry conserved domains, which suggests they may serve as effector proteins for the T6SS. A number of the proteins also show homology to the C-terminal extensions of evolved VgrG proteins. Conclusions Extensive diversity was observed in the number and content of the T6SS loci among the Pantoea and Erwinia species. Genomic islands could be observed within some of T6SS loci, which are associated with the hcp and vgrG proteins and carry putative effector domain proteins. We propose new hypotheses concerning a role for these islands in the acquisition of T6SS effectors and the development of novel evolved VgrG and Hcp proteins. PMID:22115407

  16. Comparative genomics of the Type VI secretion systems of Pantoea and Erwinia species reveals the presence of putative effector islands that may be translocated by the VgrG and Hcp proteins.

    PubMed

    De Maayer, Pieter; Venter, Stephanus N; Kamber, Tim; Duffy, Brion; Coutinho, Teresa A; Smits, Theo H M

    2011-11-24

    The Type VI secretion apparatus is assembled by a conserved set of proteins encoded within a distinct locus. The putative effector proteins Hcp and VgrG are also encoded within these loci. We have identified numerous distinct Type VI secretion system (T6SS) loci in the genomes of several ecologically diverse Pantoea and Erwinia species and detected the presence of putative effector islands associated with the hcp and vgrG genes. Between two and four T6SS loci occur among the Pantoea and Erwinia species. While two of the loci (T6SS-1 and T6SS-2) are well conserved among the various strains, the third (T6SS-3) locus is not universally distributed. Additional orthologous loci are present in Pantoea sp. aB-valens and Erwinia billingiae Eb661. Comparative analysis of the T6SS-1 and T6SS-3 loci showed non-conserved islands associated with the vgrG and hcp, and vgrG genes, respectively. These regions had a G+C content far lower than the conserved portions of the loci. Many of the proteins encoded within the hcp and vgrG islands carry conserved domains, which suggests they may serve as effector proteins for the T6SS. A number of the proteins also show homology to the C-terminal extensions of evolved VgrG proteins. Extensive diversity was observed in the number and content of the T6SS loci among the Pantoea and Erwinia species. Genomic islands could be observed within some of T6SS loci, which are associated with the hcp and vgrG proteins and carry putative effector domain proteins. We propose new hypotheses concerning a role for these islands in the acquisition of T6SS effectors and the development of novel evolved VgrG and Hcp proteins.

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

    PubMed

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

    2015-09-01

    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. The virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S substrates are being

  18. 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

  19. Odorant-binding protein: localization to nasal glands and secretions.

    PubMed Central

    Pevsner, J; Sklar, P B; Snyder, S H

    1986-01-01

    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. Images PMID:3523479

  20. Secretion and extracellular space travel of Wnt proteins.

    PubMed

    Gross, Julia Christina; Boutros, Michael

    2013-08-01

    Wnt signaling pathways control many processes during development, stem cell maintenance and homeostasis, and their aberrant regulation has been linked to diseases in man including diabetes, neurodegeneration and cancer. Wnts are hydrophobic proteins, however, quite paradoxically, they can travel over distances to induce cell-type specific responses. While there has been an initial focus on elucidating the intracellular signaling cascade, discoveries in the past few years have shed light on a highly complex, and regulated secretory process that guides Wnt proteins through the exocytic pathway. Wnt proteins are at least in portion packaged onto extracellular carriers such as exosomes. Similar to dysregulation of components in the Wnt receiving cell, failure to regulate Wnt secretion has been linked to cancer. Here, we review recent discoveries on factors and processes implicated in Wnt secretion.

  1. A novel fusion partner for enhanced secretion of recombinant proteins in Saccharomyces cerevisiae.

    PubMed

    Bae, Jung-Hoon; Sung, Bong Hyun; Seo, Jeong-Woo; Kim, Chul Ho; Sohn, Jung-Hoon

    2016-12-01

    Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.

  2. YscP of Yersinia pestis Is a Secreted Component of the Yop Secretion System

    PubMed Central

    Payne, Patricia L.; Straley, Susan C.

    1999-01-01

    The Yersinia pestis low-Ca2+ response stimulon is responsible for the environmentally regulated expression and secretion of antihost proteins (V antigen and Yops). We have previously shown that yscO encodes a secreted core component of the Yop secretion (Ysc) mechanism. In this study, we constructed and characterized in-frame deletions in the adjacent gene, yscP, in the yscN–yscU operon. The ΔP1 mutation, which removed amino acids 246 to 333 of YscP, had no effect on Yop expression or secretion, and the mutant protein, YscP1, was secreted, as was YscP in the parent. In contrast, the ΔP2 strain expressed and secreted less of each Yop than did the parent under the inductive conditions of 37°C and the absence of Ca2+, with an exception being YopE, which was only minimally affected by the mutation. The YscP2 protein, missing amino acids 57 to 324 of YscP, was expressed but not secreted by the ΔP2 mutant. The effect of the ΔP2 mutation was at the level of Yop secretion because YopM and V antigen still showed limited secretion when overproduced in trans. Excess YscP also affected secretion: overexpression of YscP in the parent, in either yscP mutant, or in an lcrG mutant effectively shut off secretion. However, co-overexpression of YscO and YscP had no effect on secretion, and YscP overexpression in an lcrE mutant had little effect on Yop secretion, suggesting that YscP acts, in conjunction with YscO, at the level of secretion control of LcrE at the bacterial surface. These findings place YscP among the growing family of mobile Ysc components that both affect secretion and themselves are secreted by the Ysc. PMID:10217778

  3. Escherichia coli-induced temporal and differential secretion of heat-shock protein 70 and interleukin-1β by human fetal membranes in a two-compartment culture system.

    PubMed

    Osorio-Caballero, M; Perdigón-Palacio, C; García-López, G; Flores-Herrera, O; Olvera-Sánchez, S; Morales-Méndez, I; Sosa-González, I; Acevedo, J F; Guzmán-Grenfell, A M; Molina-Hernández, A; Díaz, N F; Flores-Herrera, H

    2015-03-01

    Escherichia coli is recognized as an etiological bacteria associated with chorioamnionitis and the preterm premature rupture of fetal membranes. This pathological condition induces pro-inflammatory cytokines and degradative metalloproteinases, which are considered biological markers secreted in an acute stage of infection. Heat-shock proteins (HSPs) are an important component of the innate immunity response and are found in different pathological conditions. They have not been previously measured in human fetal membranes in response to infectious conditions. We hypothesized that the choriodecidual tissue and amniotic epithelium secreted temporal and differential Hsp-60, Hsp-70, and interleukin (IL)-1β mediated by E. coli infection. Fetal membranes were mounted in a two-compartment culture system and infected with two passes of live E. coli at different doses (10², 10⁴, 10⁵, and 10⁶ colony-forming units (CFU)/mL) and intervals of incubation (3, 6, and 24 h). The culture medium was collected, and Hsp-60, Hsp-70, and IL-1β were assessed using the enzyme-linked immunosorbent assay (ELISA) method. After 3 and 6 h of infection, E. coli induced an increase in Hsp-70 secretion in the choriodecidual tissue. However, after 24 h of incubation, Hsp-70 was downregulated and we observed an increase in IL-1β secretion. By contrast, E. coli induced a lower Hsp-60 secretion in the amnion compared to Hsp-70. Human fetal membranes responded actively to E. coli infection, with an increase in Hsp-70 during the first hours of infection. After 24 h, there was an increase in the liberation of IL-1β. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2014-04-18

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. 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

  6. Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition.

    PubMed

    Zhang, Ningning; Dolan, Maureen; Wu, Di; Phillips, Gregory C; Xu, Jianfeng

    2016-12-01

    Cell growth medium composition has profound impacts on the O -glycosylation of a "designer" arabinogalactan protein-based module; full glycosylation is essential in directing efficient extracellular secretion of the tagged recombinant protein. Expression of recombinant proteins in plant cells as fusion with a de novo designed hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) tag, termed HypGP engineering technology, resulted in dramatically increased secreted protein yields. This is due to the function of the HypGP tag as a molecular carrier in promoting efficient transport of conjoined proteins into culture media. To optimize the cell culture to achieve the best secreted protein yields, the medium effects on the cell growth and protein secretion were investigated using as a model system the tobacco BY-2 cell expressing enhanced green fluorescence protein (EGFP) fused with a (SP)32 tag (32 tandem repeats of "Ser-Pro" motif). The (SP)32 tag was found to undergo two-stage Hyp-O-glycosylation in plant cells with the dramatic secretion of the conjoined EGFP correlating with the triggering of the second-stage glycosylation. The BY-2 cell culture in SH medium generated a high secreted protein yield (125 mg/L) with a low cell biomass accumulation (~7.5 gDW/L). In contrast, very low secreted protein yields (~1.5 mg/L) with a high cell biomass accumulation (13.5 gDW/L) were obtained in MS medium. The macronutrients, specifically, the nitrogen supply greatly impacted the glycosylation of the (SP)32 tag and subsequent protein secretion. Modified MS medium with reduced nitrogen levels boosted the secreted EGFP yields to 168 mg/L. This study demonstrates the profound impacts of medium composition on the secreted yields of a HypGP-tagged protein, and provides a basis for medium design to achieve the highest productivity of the HypGP engineering technology.

  7. 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

  8. The Ess/Type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria

    PubMed Central

    Cao, Zhenping; Casabona, M. Guillermina; Kneuper, Holger; Chalmers, James D.; Palmer, Tracy

    2017-01-01

    Summary The type VII protein secretion system (T7SS) plays a critical role in the virulence of human pathogens including Mycobacterium tuberculosis and Staphylococcus aureus. Here we report that the S. aureus T7SS secretes a large nuclease toxin, EsaD. The toxic activity of EsaD is neutralised during its biosynthesis through complex formation with an antitoxin, EsaG, which binds to its C-terminal nuclease domain. The secretion of EsaD is dependent upon a further accessory protein, EsaE, that does not interact with the nuclease domain, but instead binds to the EsaD N-terminal region. EsaE has a dual cytoplasmic/membrane localization and membrane-bound EsaE interacts with the T7SS secretion ATPase, EssC, implicating EsaE in targeting the EsaDG complex to the secretion apparatus. EsaD and EsaE are co-secreted whereas EsaG is found only in the cytoplasm and may be stripped off during the secretion process. Strain variants of S. aureus that lack esaD encode at least two copies of EsaG-like proteins most likely to protect themselves from the toxic activity of EsaD secreted by esaD+ strains. In support of this, a strain overproducing EsaD elicits significant growth inhibition against a sensitive strain. We conclude that T7SSs may play unexpected and key roles in bacterial competitiveness. PMID:27723728

  9. Inhibition of pancreatic protein secretion by ghrelin in the rat

    PubMed Central

    Zhang, Weizhen; Chen, Min; Chen, Xuequn; Segura, Bradley J; Mulholland, Michael W

    2001-01-01

    The role of ghrelin in the regulation of pancreatic protein secretion was investigated in vivo using anaesthetized rats with pancreatic ductal cannulas, and in isolated pancreatic acinar cells and pancreatic lobules in vitro. In vivo, pancreatic protein output stimulated by CCK-8 (400 pmol kg−1 h−1) was dose-dependently inhibited by continuous ghrelin infusion (1.2 and 12 nmol kg−1 h−1) by 45 ± 8 and 84 ± 7 %, respectively. In rats with acute subdiaphragmatic vagotomy, ghrelin (12 nmol kg−1 h−1) significantly inhibited CCK-stimulated pancreatic protein secretion by 75 ± 18 %. Infusion of ghrelin (12 nmol kg−1 h−1) abolished pancreatic protein secretion caused by the central vagal stimulant 2-deoxy-d-glucose (75 mg kg−1), whereas bethanechol-stimulated pancreatic protein output was inhibited by only 59 ± 7 %. In vitro, ghrelin (10−11–10−7m) produced no change in basal amylase release from dispersed, purified acinar cells. Co-incubation of ghrelin (10−11−10−7m) with CCK−8 (10−10m) demonstrated no inhibition of CCK-stimulated amylase release from dispersed acini. In contrast, ghrelin (10−9−10−7m) dose-dependently inhibited amylase release from pancreatic lobules exposed to 75 mm potassium. Our results show that (1) ghrelin is a potent inhibitor of pancreatic exocrine secretion in anaesthetized rats in vivo and in pancreatic lobules in vitro; and (2) the actions of ghrelin are indirect and may be exerted at the level of intrapancreatic neurons. PMID:11711576

  10. YspM, a newly identified Ysa type III secreted protein of Yersinia enterocolitica.

    PubMed

    Witowski, Sarah E; Walker, Kimberly A; Miller, Virginia L

    2008-11-01

    Yersinia enterocolitica has three type three secretion systems, the flagellar, the plasmid Ysc type III secretion system (T3SS), and the chromosomal Ysa T3SS. The Ysc T3SS, through the proteins it secretes (Yops), prevents phagocytosis of Y. enterocolitica and is required for disease processes in the mouse host. Recent data demonstrate a role for the Ysa T3SS during initial colonization of the mouse via secretion of Ysps (Yersinia secreted proteins). This work characterizes the discovery of a newly identified Ysa type III secreted protein, YspM. Expression of yspM is regulated by temperature, NaCl concentration, and other known regulators of the ysa system. In addition, YspM is translocated into host cells via the Ysa T3SS. YspM is homologous to proteins classified as GDSL bacterial lipases, which possess a catalytic triad of amino acids (Ser, Asp, and His) located in three of five blocks of amino acid identity. Sequence analysis of the JB580v strain of Y. enterocolitica shows that, due to a premature stop codon, it no longer encodes the fifth block of amino acid identity containing the predicted catalytic histidine. However, seven other biotype 1B strains sequenced did possess the domain. A functional difference between the forms was revealed when YspM was expressed in Saccharomyces cerevisiae. Yeast growth was uninhibited when YspM from JB580v was expressed but greatly inhibited when YspM from Y295 (YspM(Y295)) was expressed. Site-directed mutagenesis of the histidine of YspM(Y295) ablated the toxic effects. These results indicate that YspM is secreted by the Ysa T3SS and that, possibly due to lipase activity, it targets eukaryotic cellular component(s).

  11. Structure of a Type-1 Secretion System ABC Transporter.

    PubMed

    Morgan, Jacob L W; Acheson, Justin F; Zimmer, Jochen

    2017-03-07

    Type-1 secretion systems (T1SSs) represent a widespread mode of protein secretion across the cell envelope in Gram-negative bacteria. The T1SS is composed of an inner-membrane ABC transporter, a periplasmic membrane-fusion protein, and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing a conduit for the translocation of unfolded polypeptides. We show that ATP hydrolysis and assembly of the entire T1SS complex is necessary for protein secretion. Furthermore, we present a 3.15-Å crystal structure of AaPrtD, the ABC transporter found in the Aquifex aeolicus T1SS. The structure suggests a substrate entry window just above the transporter's nucleotide binding domains. In addition, highly kinked transmembrane helices, which frame a narrow channel not observed in canonical peptide transporters, are likely involved in substrate translocation. Overall, the AaPrtD structure supports a polypeptide transport mechanism distinct from alternating access.

  12. Secreted proteins of Avibacterium paragallinarum are lethal for chicken embryo.

    PubMed

    Pérez-Márquez, Víctor; Pérez-Méndez, Alma; Ibarra-Caballero, Jorge; Gómez-Lugo, Gabriela; Vázquez-Cruz, Candelario; Vaca, Sergio; Negrete-Abascal, Erasmo

    2008-12-01

    Avibacterium paragallinarum causes infectious coryza in chickens. This bacterium secretes proteins of 110 kDa (a putative RTX protein) and 120 kDa. Expression of these proteins increases by the addition of CaCl(2), MgSO(4), MnSO(4), or ferric ammonium citrate and diminishes with CuSO(4) or ZnCl(2). Protein expression is optimal at 37 degrees C and pH 7.5. Mortality (90-100%) of chicken embryos was observed when secreted proteins (SPs) from A. paragallinarum reference or field isolates (serogroup A or C) were inoculated via yolk sac and was not observed when SPs from A. avium, a chicken respiratory tract indigenous bacterium, were inoculated. A. paragallinarum SPs could contain toxins responsible for the embryo deaths. Indeed, presence of the putative RTX protein of 110 kDa was confirmed by Western blotting with antibodies against the Actinobacillus pleuropneumoniae RTX ApxI, a closely related RTX protein.

  13. MatureP: prediction of secreted proteins with exclusive information from their mature regions.

    PubMed

    Orfanoudaki, Georgia; Markaki, Maria; Chatzi, Katerina; Tsamardinos, Ioannis; Economou, Anastassios

    2017-06-12

    More than a third of the cellular proteome is non-cytoplasmic. Most secretory proteins use the Sec system for export and are targeted to membranes using signal peptides and mature domains. To specifically analyze bacterial mature domain features, we developed MatureP, a classifier that predicts secretory sequences through features exclusively computed from their mature domains. MatureP was trained using Just Add Data Bio, an automated machine learning tool. Mature domains are predicted efficiently with ~92% success, as measured by the Area Under the Receiver Operating Characteristic Curve (AUC). Predictions were validated using experimental datasets of mutated secretory proteins. The features selected by MatureP reveal prominent differences in amino acid content between secreted and cytoplasmic proteins. Amino-terminal mature domain sequences have enhanced disorder, more hydroxyl and polar residues and less hydrophobics. Cytoplasmic proteins have prominent amino-terminal hydrophobic stretches and charged regions downstream. Presumably, secretory mature domains comprise a distinct protein class. They balance properties that promote the necessary flexibility required for the maintenance of non-folded states during targeting and secretion with the ability of post-secretion folding. These findings provide novel insight in protein trafficking, sorting and folding mechanisms and may benefit protein secretion biotechnology.

  14. 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.

  15. Secretion and Proteolysis of Heterologous Proteins Fused to the Escherichia coli Maltose Binding Protein in Pichia pastoris

    PubMed Central

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

    2010-01-01

    The E. 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

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. Pathogenic Acinetobacter Species have a Functional Type I Secretion System and Contact-Dependent Inhibition Systems.

    PubMed

    Harding, Christian M; Pulido, Marina R; Di Venanzio, Gisela; Kinsella, Rachel L; Webb, Andrew I; Scott, Nichollas E; Pachón, Jerónimo; Feldman, Mario F

    2017-04-03

    Pathogenic Acinetobacter species, including A. baumannii and A. nosocomialis, are opportunistic human pathogens of increasing relevance worldwide. Although their mechanisms of drug resistance are well studied, the virulence factors that govern Acinetobacter pathogenesis are incompletely characterized. Here we define the complete secretome of A. nosocomialis strain M2 in minimal media and demonstrate that pathogenic Acinetobacter species produce both a functional type I secretion system (T1SS) and a contact dependent inhibition (CDI) system. Using bioinformatics, quantitative proteomics, and mutational analyses we show that Acinetobacter uses its T1SS for exporting two putative T1SS effectors, an RTX-Serralysin-like toxin and the biofilm associated protein (Bap). Moreover, we found that mutation of any component of the T1SS system abrogated type VI secretion activity under nutrient-limited conditions, indicating a previously unrecognized crosstalk between these two systems. We also demonstrate that the Acinetobacter T1SS is required for biofilm formation. Lastly, we show that both A. nosocomialis and A. baumannii produce functioning CDI systems that mediate growth inhibition of sister cells lacking the cognate immunity protein. The Acinetobacter CDI systems are widely distributed across pathogenic Acinetobacter species, with many A. baumannii isolates harboring two distinct CDI systems. Collectively, these data demonstrate the power of differential, quantitative proteomics approaches to study secreted proteins, define the role of previously uncharacterized protein export systems, and observe crosstalk between secretion systems in the pathobiology of medically relevant Acinetobacter The data are available via ProteomeXchange with identifier PXD005881.

  1. Identification of lipid synthesis and secretion proteins in bovine milk.

    PubMed

    Lu, Jing; van Hooijdonk, Toon; Boeren, Sjef; Vervoort, Jacques; Hettinga, Kasper

    2014-02-01

    Lactation physiology is a process that is only partly understood. Proteomics techniques have shown to be useful to help advance the knowledge on lactation physiology in human and rodent species but have not been used as major tools for dairy cows, except for mastitis. In this paper, advanced non-targeted proteomics techniques (Filter aided sample preparation and NanoLC-Orbitrap-MS/MS) were applied to study the milk fat globule membrane and milk serum fraction, resulting in the identification of 246 proteins. Of these, 23 transporters and enzymes were related to lipid synthesis and secretion in mammary gland and their functions are discussed in detail. The identification of these intracellular transporters and enzymes in milk provides a possibility of using milk itself to study lipid synthesis and secretion pathways. This full-scale scan of milk proteins by using non-targeted proteomic analysis helps to reveal the important proteins involved in lipid synthesis and secretion for further examination in targeted studies.

  2. 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

  3. 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

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

    DOE PAGES

    Fan, Yao; Tan, Kemin; Chhor, Gekleng; ...

    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

  5. 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

    2015-09-01

    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.

  6. 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

  7. The level of Yop proteins secreted by Yersinia enterocolitica is changed in maltose mutants.

    PubMed

    Brzostek, K; Raczkowska, A

    2001-10-16

    Enteropathogenic Yersinia enterocolitica strains express a set of plasmid-encoded proteins called Yops, involved in pathogenicity. We studied the influence of the maltose system on the production of Yop proteins and found that the level of Yop proteins of Y. enterocolitica O:9 was reduced in the presence of maltose. Transposon insertion mutants impaired with the maltose transport activity showed a decreased level in the production of Yop proteins. The transcription of the yopH gene for YopH phosphatase in these maltose mutants was unchanged and revealed a maltose mutation impaired in the secretion of Yop proteins instead of their expression.

  8. Type Three Secretion System in Attaching and Effacing Pathogens.

    PubMed

    Gaytán, Meztlli O; Martínez-Santos, Verónica I; Soto, Eduardo; González-Pedrajo, Bertha

    2016-01-01

    Enteropathogenic Escherichia coli and enterohemorrhagic E. coli are diarrheagenic bacterial human pathogens that cause severe gastroenteritis. These enteric pathotypes, together with the mouse pathogen Citrobacter rodentium, belong to the family of attaching and effacing pathogens that form a distinctive histological lesion in the intestinal epithelium. The virulence of these bacteria depends on a type III secretion system (T3SS), which mediates the translocation of effector proteins from the bacterial cytosol into the infected cells. The core architecture of the T3SS consists of a multi-ring basal body embedded in the bacterial membranes, a periplasmic inner rod, a transmembrane export apparatus in the inner membrane, and cytosolic components including an ATPase complex and the C-ring. In addition, two distinct hollow appendages are assembled on the extracellular face of the basal body creating a channel for protein secretion: an approximately 23 nm needle, and a filament that extends up to 600 nm. This filamentous structure allows these pathogens to get through the host cells mucus barrier. Upon contact with the target cell, a translocation pore is assembled in the host membrane through which the effector proteins are injected. Assembly of the T3SS is strictly regulated to ensure proper timing of substrate secretion. The different type III substrates coexist in the bacterial cytoplasm, and their hierarchical secretion is determined by specialized chaperones in coordination with two molecular switches and the so-called sorting platform. In this review, we present recent advances in the understanding of the T3SS in attaching and effacing pathogens.

  9. Type Three Secretion System in Attaching and Effacing Pathogens

    PubMed Central

    Gaytán, Meztlli O.; Martínez-Santos, Verónica I.; Soto, Eduardo; González-Pedrajo, Bertha

    2016-01-01

    Enteropathogenic Escherichia coli and enterohemorrhagic E. coli are diarrheagenic bacterial human pathogens that cause severe gastroenteritis. These enteric pathotypes, together with the mouse pathogen Citrobacter rodentium, belong to the family of attaching and effacing pathogens that form a distinctive histological lesion in the intestinal epithelium. The virulence of these bacteria depends on a type III secretion system (T3SS), which mediates the translocation of effector proteins from the bacterial cytosol into the infected cells. The core architecture of the T3SS consists of a multi-ring basal body embedded in the bacterial membranes, a periplasmic inner rod, a transmembrane export apparatus in the inner membrane, and cytosolic components including an ATPase complex and the C-ring. In addition, two distinct hollow appendages are assembled on the extracellular face of the basal body creating a channel for protein secretion: an approximately 23 nm needle, and a filament that extends up to 600 nm. This filamentous structure allows these pathogens to get through the host cells mucus barrier. Upon contact with the target cell, a translocation pore is assembled in the host membrane through which the effector proteins are injected. Assembly of the T3SS is strictly regulated to ensure proper timing of substrate secretion. The different type III substrates coexist in the bacterial cytoplasm, and their hierarchical secretion is determined by specialized chaperones in coordination with two molecular switches and the so-called sorting platform. In this review, we present recent advances in the understanding of the T3SS in attaching and effacing pathogens. PMID:27818950

  10. Patterns of gene expression in pig adipose tissue: insulin-like growth factor system proteins, neuropeptide Y (NPY), NPY receptors, neurotrophic factors and other secreted factors.

    PubMed

    Hausman, G J; Barb, C R; Dean, R G

    2008-07-01

    Although cDNA microarray studies have examined gene expression in human and rodent adipose tissue, only one microarray study of adipose tissue from growing pigs has been reported. Total RNA was collected at slaughter from outer subcutaneous adipose tissue (OSQ) and middle subcutaneous adipose tissue (MSQ) from gilts at 90, 150, and 210 d (n=5 age(-1)). Dye labeled cDNA probes were hybridized to custom porcine microarrays (70-mer oligonucleotides). Gene expression of insulin-like growth factor binding proteins (IGFBPs), hormones, growth factors, neuropeptide Y (NPY) receptors (NPYRs) and other receptors in OSQ and MSQ changed little with age in growing pigs. Distinct patterns of relative gene expression were evident within NPYR and IGFBP family members in adipose tissue from growing pigs. Relative gene expression levels of NPY2R, NPY4R and angiopoietin 2 (ANG-2) distinguished OSQ and MSQ depots in growing pigs. We demonstrated, for the first time, the expression of IGFBP-7, IGFBP-5, NPY1R, NPY2R, NPY, connective tissue growth factor (CTGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) genes in pig adipose tissue with microarray and RT-PCR assays. Furthermore, adipose tissue CTGF gene expression was upregulated while NPY and NPY2R gene expression were significantly down regulated by age. These studies demonstrate that expression of neuropeptides and neurotrophic factors in pig adipose tissue may be involved in regulation of leptin secretion. Many other regulatory factors were not influenced by age in growing pigs but may be influenced by location or depot.

  11. Adaptive Evolution in Rodent Seminal Vesicle Secretion Proteins

    PubMed Central

    Clark, Nathaniel L.; Nguyen, Eric D.; Swanson, Willie J.

    2008-01-01

    Proteins involved in reproductive fitness have evolved unusually rapidly across diverse groups of organisms. These reproductive proteins show unusually high rates of amino acid substitutions, suggesting that the proteins have been subject to positive selection. We sought to identify seminal fluid proteins experiencing adaptive evolution because such proteins are often involved in sperm competition, host immunity to pathogens, and manipulation of female reproductive physiology and behavior. We performed an evolutionary screen of the mouse prostate transcriptome for genes with elevated evolutionary rates between mouse and rat. We observed that secreted rodent prostate proteins evolve approximately twice as fast as nonsecreted proteins, remarkably similar to findings in the primate prostate and in the Drosophila male accessory gland. Our screen led us to identify and characterize a group of seminal vesicle secretion (Svs) proteins and to show that the gene Svs7 is evolving very rapidly, with many amino acid sites under positive selection. Another gene in this group, Svs5, showed evidence of branch-specific selection in the rat. We also found that Svs7 is under selection in primates and, by using three-dimensional models, demonstrated that the same regions have been under selection in both groups. Svs7 has been identified as mouse caltrin, a protein involved in sperm capacitation, the process responsible for the timing of changes in sperm activity and behavior, following ejaculation. We propose that the most likely explanation of the adaptive evolution of Svs7 that we have observed in rodents and primates stems from an important function in sperm competition. PMID:18718917

  12. Distribution of Secretion Systems in the Genus Legionella and Its Correlation with Pathogenicity

    PubMed Central

    Qin, Tian; Zhou, Haijian; Ren, Hongyu; Liu, Wenbin

    2017-01-01

    The genus Legionella comprises over 60 species, which are important human pathogens. Secretion systems in Legionella pneumophila have been studied extensively because of the essential role of protein secretion in bacterial infection. However, there are few reports describing the secretion systems in non-L. pneumophila species. In this study, we analyzed the distribution of secretion systems in L. pneumophila and 18 species of non-L. pneumophila based on whole genome sequences. A total of 74 whole genome sequences from 19 species of Legionella were analyzed. Type II and IVB secretion systems were detected in all Legionella strains, but the type I secretion systems was restricted to L. pneumophila. The type IVA secretion system was randomly distributed among different species. Furthermore, we found the type VI secretion system in three non-L. pneumophila strains (Legionella cherrii DSM 19213, Legionella dumoffii Tex-KL, and Legionella gormanii ATCC 33297). In population structure analysis, L. pneumophila formed a conservative cluster and was located at the terminal of the evolutionary tree. At the same time, L. pneumophila, especially eight clone groups (named MCGG1–MCGG8), showed higher intracellular growth ability than non-L. pneumophila species. These results suggest that L. pneumophila has acquired additional secretion systems during evolution, resulting in increased pathogenicity. PMID:28352254

  13. Angiotensin II-Activated Protein Kinase D Mediates Acute Aldosterone Secretion

    PubMed Central

    Shapiro, Brian A.; Olala, Lawrence; Arun, Senthil Nathan; Parker, Peter M.; George, Mariya V.; Bollag, Wendy B.

    2009-01-01

    Summary Dysregulation of the renin-angiotensin II (AngII)-aldosterone system can contribute to cardiovascular disease, such that an understanding of this system is critical. Diacylglycerol-sensitive serine/threonine protein kinase D (PKD) is activated by AngII in several systems, including the human adrenocortical carcinoma cell line NCI H295R, where this enzyme enhances chronic (24 hours) AngII-evoked aldosterone secretion. However, the role of PKD in acute AngII-elicited aldosterone secretion has not been previously examined. In primary cultures of bovine adrenal glomerulosa cells, which secrete detectable quantities of aldosterone in response to secretagogues within minutes, PKD was activated in response to AngII, but not an elevated potassium concentration or adrenocorticotrophic hormone. This activation was time- and dose-dependent and occurred through the AT1, but not the AT2, receptor. Adenovirus-mediated overexpression of constitutively-active PKD resulted in enhanced AngII-induced aldosterone secretion; whereas overexpression of a dominant-negative PKD construct decreased AngII-stimulated aldosterone secretion. Thus, we demonstrate for the first time that PKD mediates acute AngII-induced aldosterone secretion. PMID:19961896

  14. Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein.

    PubMed

    Liu, J; Wang, Y; Szalay, A A; Escher, A

    2000-01-01

    We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells.

  15. Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

    DOE PAGES

    Ekiert, Damian C.; Cox, Jeffery S.

    2014-10-01

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed "type VII." How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here in this paper, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), whichmore » adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE-PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Furthermore, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.« less

  16. Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion.

    PubMed

    Ekiert, Damian C; Cox, Jeffery S

    2014-10-14

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed "type VII." How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE-PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.

  17. 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

  18. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties.

    PubMed

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

    2016-05-30

    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.

  19. Secretion and transfer of the thyroid hormone binding protein transthyretin by human placenta.

    PubMed

    Mortimer, R H; Landers, K A; Balakrishnan, B; Li, H; Mitchell, M D; Patel, J; Richard, K

    2012-04-01

    The thyroid hormone and retinol binding protein transthyretin (TTR) is synthesised by human trophoblasts. Polarised JEG-3 choriocarcinoma cells grown in bicameral chambers secrete TTR predominantly apically but also basally and these cells and human trophoblasts also take up TTR suggesting that there may be a placental TTR shuttle that participates in materno-fetal transfer of thyroid hormones and retinol. Our objective was to investigate TTR secretion into the maternal and fetal circuits of the ex vivo dually perfused placental lobule to confirm that placenta secretes TTR into the fetal circulation. We also investigated translocation of Alexa Fluor-594 labelled TTR from incubation medium into the fetal placental capillaries in early (14-15 weeks) and term placental villus explants. The perfused placental lobule secretes TTR into the maternal and fetal circuits. Secretion in both circuits is linear with time and is predominantly into the maternal circuit (mean maternal/fetal ratio 99.4 ± 25.6). The mean data fitted well to a three compartment mathematical model (maternal circuit, placenta and fetal circuit, constant secretion of TTR and return of maternal circuit TTR to the placental compartment). Explants from early (14-15 weeks) and late (38-40 weeks) placentas translocated fluorescently labelled TTR from medium to villus (fetal) capillaries. Our results confirm that human placenta secretes TTR into maternal and fetal circulations and supports the hypothesis that placental TTR secreted into the maternal placental circulation can be taken up by trophoblasts and translocated to the fetal circulation, forming a TTR shuttle system. This may have important implications for materno-fetal transfer of thyroid hormones, retinol/retinol binding protein and xenobiotics (such as polychlorinated biphenyls) all of which bind to TTR. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  20. Modulation of host immune defenses by Aeromonas and Yersinia species: convergence on toxins secreted by various secretion systems.

    PubMed

    Rosenzweig, Jason A; Chopra, Ashok K

    2013-01-01

    Like other pathogenic bacteria, Yersinia and Aeromonas species have been continuously co-evolving with their respective hosts. Although the former is a bonafide human pathogen, the latter has gained notararity as an emerging disease-causing agent. In response to immune cell challenges, bacterial pathogens have developed diverse mechanism(s) enabling their survival, and, at times, dominance over various host immune defense systems. The bacterial type three secretion system (T3SS) is evolutionarily derived from flagellar subunits and serves as a vehicle by which microbes can directly inject/translocate anti-host factors/effector proteins into targeted host immune cells. A large number of Gram-negative bacterial pathogens possess a T3SS empowering them to disrupt host cell signaling, actin cytoskeleton re-arrangements, and even to induce host-cell apoptotic and pyroptotic pathways. All pathogenic yersiniae and most Aeromonas species possess a T3SS, but they also possess T2- and T6-secreted toxins/effector proteins. This review will focus on the mechanisms by which the T3SS effectors Yersinia outer membrane protein J (YopJ) and an Aeromonas hydrophila AexU protein, isolated from the diarrheal isolate SSU, mollify host immune system defenses. Additionally, the mechanisms that are associated with host cell apoptosis/pyroptosis by Aeromonas T2SS secreted Act, a cytotoxic enterotoxin, and Hemolysin co-regulated protein (Hcp), an A. hydrophila T6SS effector, will also be discussed.

  1. 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.

  2. 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

  3. Mechanism and structure of the bacterial type IV secretion systems.

    PubMed

    Christie, Peter J; Whitaker, Neal; González-Rivera, Christian

    2014-08-01

    The bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to bacterial or eukaryotic target cells generally by a mechanism dependent on direct cell-to-cell contact. The T4SSs encompass two large subfamilies, the conjugation systems and the effector translocators. The conjugation systems mediate interbacterial DNA transfer and are responsible for the rapid dissemination of antibiotic resistance genes and virulence determinants in clinical settings. The effector translocators are used by many Gram-negative bacterial pathogens for delivery of potentially hundreds of virulence proteins to eukaryotic cells for modulation of different physiological processes during infection. Recently, there has been considerable progress in defining the structures of T4SS machine subunits and large machine subassemblies. Additionally, the nature of substrate translocation sequences and the contributions of accessory proteins to substrate docking with the translocation channel have been elucidated. A DNA translocation route through the Agrobacterium tumefaciens VirB/VirD4 system was defined, and both intracellular (DNA ligand, ATP energy) and extracellular (phage binding) signals were shown to activate type IV-dependent translocation. Finally, phylogenetic studies have shed light on the evolution and distribution of T4SSs, and complementary structure-function studies of diverse systems have identified adaptations tailored for novel functions in pathogenic settings. This review summarizes the recent progress in our understanding of the architecture and mechanism of action of these fascinating machines, with emphasis on the 'archetypal' A. tumefaciens VirB/VirD4 T4SS and related conjugation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Conjugative type IV secretion systems in Gram-positive bacteria.

    PubMed

    Goessweiner-Mohr, Nikolaus; Arends, Karsten; Keller, Walter; Grohmann, Elisabeth

    2013-11-01

    Bacterial conjugation presents the most important means to spread antibiotic resistance and virulence factors among closely and distantly related bacteria. Conjugative plasmids are the mobile genetic elements mainly responsible for this task. All the genetic information required for the horizontal transmission is encoded on the conjugative plasmids themselves. Two distinct concepts for horizontal plasmid transfer in Gram-positive bacteria exist, the most prominent one transports single stranded plasmid DNA via a multi-protein complex, termed type IV secretion system, across the Gram-positive cell envelope. Type IV secretion systems have been found in virtually all unicellular Gram-positive bacteria, whereas multicellular Streptomycetes seem to have developed a specialized system more closely related to the machinery involved in bacterial cell division and sporulation, which transports double stranded DNA from donor to recipient cells. This review intends to summarize the state of the art of prototype systems belonging to the two distinct concepts; it focuses on protein key players identified so far and gives future directions for research in this emerging field of promiscuous interbacterial transport.

  5. The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis.

    PubMed

    Cosme, Ana M; Becker, Anke; Santos, Mário R; Sharypova, Larissa A; Santos, Pedro M; Moreira, Leonilde M

    2008-07-01

    Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation relationship with Medicago sativa. During this process, it must cope with diverse environments and has evolved different types of transport systems that help its propagation in the plant roots. TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In this work, we have characterized the protein TolC from S. meliloti 2011. An insertional mutation in the tolC gene strongly affected the resistance phenotype to antimicrobial agents and induced higher susceptibility to osmotic and oxidative stresses. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type versus tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of open reading frame SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by a TolC-dependent secretion system. In the absence of TolC, neither succinoglycan nor galactoglucan were detected in the culture supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of nonfixing nitrogen nodules in M. sativa roots. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.

  6. A secreted protein promotes cleavage furrow maturation during cytokinesis.

    PubMed

    Xu, Xuehong; Vogel, Bruce E

    2011-01-25

    Developmental modifications in cell shape depend on dynamic interactions between the extracellular matrix and cytoskeleton. In contrast, existing models of cytokinesis describe substantial cell surface remodeling that involves many intracellular regulatory and structural proteins but includes no contribution from the extracellular matrix [1-3]. Here, we show that extracellular hemicentins assemble at the cleavage furrow of dividing cells in the C. elegans germline and in preimplantation mouse embryos. In the absence of hemicentin, cleavage furrows form but retract prior to completion, resulting in multinucleate cells. In addition to their role in tissue organization, the data indicate that hemicentins are the first secreted proteins required during mammalian development and the only known secreted proteins required for cytokinesis, with an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows. Together with studies showing that extracellular polysaccharides are required for cytokinesis in diverse species [4-9], our data suggest that assembly of a cell type-specific extracellular matrix may be a general requirement for cleavage furrow maturation and contractile ring function during cytokinesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. A Secreted Protein Promotes Cleavage Furrow Maturation during Cytokinesis

    PubMed Central

    Xu, Xuehong; Vogel, Bruce E.

    2010-01-01

    Summary Developmental modifications in cell shape depend on dynamic interactions between the extracellular matrix and cytoskeleton. In contrast, existing models of cytokinesis describe substantial cell surface remodeling that involves many intracellular regulatory and structural proteins but includes no contribution from the extracellular matrix [1–3]. Here, we show that extracellular hemicentins assemble at the cleavage furrow of dividing cells in the C. elegans germline and in preimplantation mouse embryos. In the absence of hemicentin, cleavage furrows form but retract prior to completion, resulting in multinucleate cells. In addition to their role in tissue organization, the data indicate that hemicentins are the first secreted proteins required during mammalian development and the only known secreted proteins required for cytokinesis, with an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows. Together with studies showing that extracellular polysaccharides are required for cytokinesis in diverse species [4–9], our data suggest that assembly of a cell type-specific extracellular matrix may be a general requirement for cleavage furrow maturation and contractile ring function during cytokinesis. PMID:21215633

  8. Urothelial function reconsidered: A role in urinary protein secretion

    PubMed Central

    Deng, Fang-Ming; Ding, Mingxiao; Lavker, Robert M.; Sun, Tung-Tien

    2001-01-01

    Mammalian bladder epithelium functions as an effective permeability barrier. We demonstrate here that this epithelium can also function as a secretory tissue directly involved in modifying urinary protein composition. Our data indicate that normal bovine urothelium synthesizes, as its major differentiation products, two well-known proteases: tissue-type plasminogen activator and urokinase, as well as a serine protease inhibitor, PP5. Moreover, we demonstrate that the urothelium secretes these proteins in a polarized fashion into the urine via a cAMP- and calcium-regulated pathway. Urinary plasminogen activators of ruminants are therefore urothelium derived rather then kidney derived as in some other species; this heterogeneity may have evolved in response to different physiological or dietary factors. In conjunction with our recent finding that transgenic mouse urothelium can secrete ectopically expressed human growth hormone into the urine, our data establish that normal mammalian urothelium can function not only as a permeability barrier but also as a secretor of urinary proteins that can play physiological or pathological roles in the urinary tract. PMID:11136252

  9. Secreted protein kinases regulate cyst burden during chronic toxoplasmosis.

    PubMed

    Jones, Nathaniel G; Wang, Qiuling; Sibley, L David

    2017-02-01

    Toxoplasma gondii is an apicomplexan parasite that secretes a large number of protein kinases and pseudokinases from its rhoptry organelles. Although some rhoptry kinases (ROPKs) act as virulence factors, many remain uncharacterized. In this study, predicted ROPKs were assessed for bradyzoite expression then prioritized for a reverse genetic analysis in the type II strain Pru that is amenable to targeted disruption. Using CRISPR/Cas9, we engineered C-terminally epitope tagged ROP21 and ROP27 and demonstrated their localization to the parasitophorous vacuole and cyst matrix. ROP21 and ROP27 were not secreted from microneme, rhoptry, or dense granule organelles, but rather were located in small vesicles consistent with a constitutive pathway. Using CRISPR/Cas9, the genes for ROP21, ROP27, ROP28, and ROP30 were deleted individually and in combination, and the mutant parasites were assessed for growth and their ability to form tissue cysts in mice. All knockouts lines were normal for in vitro growth and bradyzoite differentiation, but a combined ∆rop21/∆rop17 knockout led to a 50% reduction in cyst burden in vivo. Our findings question the existing annotation of ROPKs based solely on bioinformatic techniques and yet highlight the importance of secreted kinases in determining the severity of chronic toxoplasmosis.

  10. 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.

  11. Steps for Shigella Gatekeeper Protein MxiC Function in Hierarchical Type III Secretion Regulation*

    PubMed Central

    Roehrich, A. Dorothea; Bordignon, Enrica; Mode, Selma; Shen, Da-Kang; Liu, Xia; Pain, Maria; Murillo, Isabel; Martinez-Argudo, Isabel; Sessions, Richard B.

    2017-01-01

    Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions. PMID:27974466

  12. Secretion Systems Used by Bacteria to Subvert Host Functions.

    PubMed

    Rapisarda, Chiara; Fronzes, Rémi

    2017-09-06

    In this review we examine the use of secretion systems by bacteria to subvert host functions. Bacteria have evolved multiple systems to interact with and overcome their eukaryotic host and other prokaryotes. Secretion systems are required for the release of several effectors through the bacterial membrane(s) into the extracellular space or directly into the cytoplasm of the host. We review the secretion systems of Gram-positive and Gram-negative bacteria and describe briefly the structural composition of the seven secretion systems that have been associated with increased virulence through subversion of host functions. Some of the effects of such systems on eukaryotic host processes have been studied extensively. We also describe the best-characterized effectors of each secretion system to give an overview of the molecular mechanisms employed by bacteria to hide from the immune system and convert eukaryotic cells into optimal ecological niches for their replication.

  13. Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations: How bacterial secretion systems bias Brownian motion for efficient translocation of macromolecules.

    PubMed

    Hepp, Christof; Maier, Berenike

    2017-10-01

    Secretion systems enable bacteria to import and secrete large macromolecules including DNA and proteins. While most components of these systems have been identified, the molecular mechanisms of macromolecular transport remain poorly understood. Recent findings suggest that various bacterial secretion systems make use of the translocation ratchet mechanism for transporting polymers across the cell envelope. Translocation ratchets are powered by chemical potential differences generated by concentration gradients of ions or molecules that are specific to the respective secretion systems. Bacteria employ these potential differences for biasing Brownian motion of the macromolecules within the conduits of the secretion systems. Candidates for this mechanism include DNA import by the type II secretion/type IV pilus system, DNA export by the type IV secretion system, and protein export by the type I secretion system. Here, we propose that these three secretion systems employ different molecular implementations of the translocation ratchet mechanism. © 2017 The Authors. BioEssays Published by WILEY Periodicals, Inc.

  14. THE SECRETION OF DIGESTIVE ENZYMES AND CAECAL SIZE ARE DETERMINED BY DIETARY PROTEIN IN THE CRICKET Gryllus bimaculatus.

    PubMed

    Woodring, Joseph; Weidlich, Sandy

    2016-11-01

    In Gryllus bimaculatus, the size of the caecum decreases in the latter half of each instar to a stable minimal size with a steady minimal rate of digestive enzyme secretion until feeding resumes after ecdysis. The higher the percent protein in the newly ingested food, the faster and larger the caecum grows, and as a consequent the higher the secretion rate of trypsin and amylase. When hard boiled eggs (40% protein) are eaten the caecum is 2× larger, the trypsin secretion is almost 3× greater, and amylase 2.5× greater then when fed the same amount of apples (1.5% protein). Only dietary protein increases amylase secretion, whereas dietary carbohydrates have no effect on amylase secretion. The minimal caecal size and secretion rate must be supported by utilization of hemolymph amino acids, but the growth of the caecum and increasing enzymes secretions after the molt depend upon an amino acid source in the lumen. This simple regulation of digestive enzyme secretion is ideal for animals that must stop feeding in order to molt. This basic control system does not preclude additional regulation mechanisms, such as prandal, which is also indicated for G. bimaculatus, or even paramonal regulation. © 2016 Wiley Periodicals, Inc.

  15. 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

  16. Secreted Listeria adhesion protein (Lap) influences Lap-mediated Listeria monocytogenes paracellular translocation through epithelial barrier.

    PubMed

    Kim, Hyochin; Bhunia, Arun K

    2013-06-24

    Listeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown. Amounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells. Western blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P < 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates. This study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic

  17. Secreted Listeria adhesion protein (Lap) influences Lap-mediated Listeria monocytogenes paracellular translocation through epithelial barrier

    PubMed Central

    2013-01-01

    Background Listeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown. Methods Amounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells. Results Western blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P < 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates. Conclusion This study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may

  18. 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

  19. The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function.

    PubMed

    Kim, Jinoh; Thanabalasuriar, Ajitha; Chaworth-Musters, Tessa; Fromme, J Chris; Frey, Elizabeth A; Lario, Paula I; Metalnikov, Pavel; Rizg, Keyrillos; Thomas, Nikhil A; Lee, Sau Fung; Hartland, Elizabeth L; Hardwidge, Philip R; Pawson, Tony; Strynadka, Natalie C; Finlay, B Brett; Schekman, Randy; Gruenheid, Samantha

    2007-09-13

    Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) maintain an extracellular lifestyle and use a type III secretion system to translocate effector proteins into the host cytosol. These effectors manipulate host pathways to favor bacterial replication and survival. NleA is an EHEC/EPEC- and related species-specific translocated effector protein that is essential for bacterial virulence. However, the mechanism by which NleA impacts virulence remains undetermined. Here we demonstrate that NleA compromises the Sec23/24 complex, a component of the mammalian COPII protein coat that shapes intracellular protein transport vesicles, by directly binding Sec24. Expression of an NleA-GFP fusion protein reduces the efficiency of cellular secretion by 50%, and secretion is inhibited in EPEC-infected cells. Direct biochemical experiments show that NleA inhibits COPII-dependent protein export from the endoplasmic reticulum. Collectively, these findings indicate that disruption of COPII function in host cells contributes to the virulence of EPEC and EHEC.

  20. 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

  1. Type-IVC Secretion System: A Novel Subclass of Type IV Secretion System (T4SS) Common Existing in Gram-Positive Genus Streptococcus

    PubMed Central

    Chen, Chen; Gao, George F.

    2012-01-01

    A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. Type IV secretion system (T4SS) is one of versatile secretion systems essential for the virulence and even survival of some bacteria species, and they enable the secretion of protein and DNA substrates across the cell envelope. T4SS was once believed to be present only in Gram-negative bacteria. In this study, we present evidence of a new subclass of T4SS, Type-IVC secretion system and indicate its common existence in the Gram-positive bacterial genus Streptococcus. We further identified that VirB1, VirB4, VirB6 and VirD4 are the minimal key components of this system. Using genome comparisons and evolutionary relationship analysis, we proposed that Type-IVC secretion system is movable via transposon factors and mediates the conjugative transfer of DNA, enhances bacterial pathogenicity, and could cause large-scale outbreaks of infections in humans. PMID:23056296

  2. DBSecSys 2.0: a database of Burkholderia mallei and Burkholderia pseudomallei secretion systems.

    PubMed

    Memišević, Vesna; Kumar, Kamal; Zavaljevski, Nela; DeShazer, David; Wallqvist, Anders; Reifman, Jaques

    2016-09-20

    Burkholderia mallei and B. pseudomallei are the causative agents of glanders and melioidosis, respectively, diseases with high morbidity and mortality rates. B. mallei and B. pseudomallei are closely related genetically; B. mallei evolved from an ancestral strain of B. pseudomallei by genome reduction and adaptation to an obligate intracellular lifestyle. Although these two bacteria cause different diseases, they share multiple virulence factors, including bacterial secretion systems, which represent key components of bacterial pathogenicity. Despite recent progress, the secretion system proteins for B. mallei and B. pseudomallei, their pathogenic mechanisms of action, and host factors are not well characterized. We previously developed a manually curated database, DBSecSys, of bacterial secretion system proteins for B. mallei. Here, we report an expansion of the database with corresponding information about B. pseudomallei. DBSecSys 2.0 contains comprehensive literature-based and computationally derived information about B. mallei ATCC 23344 and literature-based and computationally derived information about B. pseudomallei K96243. The database contains updated information for 163 B. mallei proteins from the previous database and 61 additional B. mallei proteins, and new information for 281 B. pseudomallei proteins associated with 5 secretion systems, their 1,633 human- and murine-interacting targets, and 2,400 host-B. mallei interactions and 2,286 host-B. pseudomallei interactions. The database also includes information about 13 pathogenic mechanisms of action for B. mallei and B. pseudomallei secretion system proteins inferred from the available literature or computationally. Additionally, DBSecSys 2.0 provides details about 82 virulence attenuation experiments for 52 B. mallei secretion system proteins and 98 virulence attenuation experiments for 61 B. pseudomallei secretion system proteins. We updated the Web interface and data access layer to speed-up users

  3. Structural studies of the Hrp secretion system: expression, purification, crystallization and preliminary X-ray analysis of the C-terminal domain of the HrcQB protein from Pseudomonas syringae pv. phaseolicola.

    PubMed

    Fadouloglou, V E; Tampakaki, A P; Panopoulos, N J; Kokkinidis, M

    2001-11-01

    The C-terminal domain of the HrcQ(B) protein from the Hrp secretion system of the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola has been crystallized from MPD using the hanging-drop vapour-diffusion method. The crystals belong to space group P2(1), with unit-cell parameters a = 51.6, b = 27.3, c = 97.2 A and beta = 99.8 degrees. A complete native data set extending to 3.0 A resolution was collected from a single cryoprotected crystal. The crystal solvent content and calculation of self-rotation functions showing non-crystallographic twofold symmetry axes are consistent with the presence of an oligomeric assembly in the asymmetric unit.

  4. 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

  5. Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa

    SciTech Connect

    Klinefelter, G.R.; Hamilton, D.W.

    1985-11-01

    We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-(35S)methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-(35S)methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB(3H)4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis.

  6. 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

  7. Gliding motility and Por secretion system genes are widespread among members of the phylum bacteroidetes.

    PubMed

    McBride, Mark J; 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.

  8. 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.

  9. 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

    2011-03-29

    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.

  10. Named Entity Recognition for Bacterial Type IV Secretion Systems

    PubMed Central

    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

  11. Heterologous Protein Secretion in Lactobacilli with Modified pSIP Vectors

    PubMed Central

    Karlskås, Ingrid Lea; Maudal, Kristina; Axelsson, Lars; Rud, Ida; Eijsink, Vincent G. H.; Mathiesen, Geir

    2014-01-01

    We describe new variants of the modular pSIP-vectors for inducible gene expression and protein secretion in lactobacilli. The basic functionality of the pSIP system was tested in Lactobacillus strains representing 14 species using pSIP411, which harbors the broad-host-range Lactococcus lactis SH71rep replicon and a β-glucuronidase encoding reporter gene. In 10 species, the inducible gene expression system was functional. Based on these results, three pSIP vectors with different signal peptides were modified by replacing their narrow-host-range L. plantarum 256rep replicon with SH71rep and transformed into strains of five different species of Lactobacillus. All recombinant strains secreted the target protein NucA, albeit with varying production levels and secretion efficiencies. The Lp_3050 derived signal peptide generally resulted in the highest levels of secreted NucA. These modified pSIP vectors are useful tools for engineering a wide variety of Lactobacillus species. PMID:24614815

  12. A systematic analysis of the RNA-targeting potential of secreted bacterial effector proteins.

    PubMed

    Tawk, Caroline; Sharan, Malvika; Eulalio, Ana; Vogel, Jörg

    2017-08-24

    Many pathogenic bacteria utilize specialized secretion systems to deliver proteins called effectors into eukaryotic cells for manipulation of host pathways. The vast majority of known effector targets are host proteins, whereas a potential targeting of host nucleic acids remains little explored. There is only one family of effectors known to target DNA directly, and effectors binding host RNA are unknown. Here, we take a two-pronged approach to search for RNA-binding effectors, combining biocomputational prediction of RNA-binding domains (RBDs) in a newly assembled comprehensive dataset of bacterial secreted proteins, and experimental screening for RNA binding in mammalian cells. Only a small subset of effectors were predicted to carry an RBD, indicating that if RNA targeting was common, it would likely involve new types of RBDs. Our experimental evaluation of effectors with predicted RBDs further argues for a general paucity of RNA binding activities amongst bacterial effectors. We obtained evidence that PipB2 and Lpg2844, effector proteins of Salmonella and Legionella species, respectively, may harbor novel biochemical activities. Our study presenting the first systematic evaluation of the RNA-targeting potential of bacterial effectors offers a basis for discussion of whether or not host RNA is a prominent target of secreted bacterial proteins.

  13. New players in the same old game: a system level in silico study to predict type III secretion system and effector proteins in bacterial genomes reveals common themes in T3SS mediated pathogenesis.

    PubMed

    Sadarangani, Vineet; Datta, Sunando; Arunachalam, Manonmani

    2013-07-26

    Type III secretion system (T3SS) plays an important role in virulence or symbiosis of many pathogenic or symbiotic bacteria [CHM 2:291-294, 2007; Physiology (Bethesda) 20:326-339, 2005]. T3SS acts like a tunnel between a bacterium and its host through which the bacterium injects 'effector' proteins into the latter [Nature 444:567-573, 2006; COSB 18:258-266, 2008]. The effectors spatially and temporally modify the host signalling pathways [FEMS Microbiol Rev 35:1100-1125, 2011; Cell Host Microbe5:571-579, 2009]. In spite its crucial role in host-pathogen interaction, the study of T3SS and the associated effectors has been limited to a few bacteria [Cell Microbiol 13:1858-1869, 2011; Nat Rev Microbiol 6:11-16, 2008; Mol Microbiol 80:1420-1438, 2011]. Before one set out to perform systematic experimental studies on an unknown set of bacteria it would be beneficial to identify the potential candidates by developing an in silico screening algorithm. A system level study would also be advantageous over traditional laboratory methods to extract an overriding theme for host-pathogen interaction, if any, from the vast resources of data generated by sequencing multiple bacterial genomes. We have developed an in silico protocol in which the most conserved set of T3SS proteins was used as the query against the entire bacterial database with increasingly stringent search parameters. It enabled us to identify several uncharacterized T3SS positive bacteria. We adopted a similar strategy to predict the presence of the already known effectors in the newly identified T3SS positive bacteria. The huge resources of biochemical data [FEMS Microbiol Rev 35:1100-1125, 2011; Cell Host Microbe 5:571-579, 2009; BMC Bioinformatics 7(11):S4, 2010] on the T3SS effectors enabled us to search for the common theme in T3SS mediated pathogenesis. We identified few cellular signalling networks in the host, which are manipulated by most of the T3SS containing pathogens. We went on to look for

  14. The plant apoplasm is an important recipient compartment for nematode secreted proteins

    PubMed Central

    Vieira, Paulo; Danchin, Etienne G. J.; Neveu, Cédric; Crozat, Carine; Jaubert, Stéphanie; Hussey, Richard S.; Engler, Gilbert; Abad, Pierre; de Almeida-Engler, Janice; Castagnone-Sereno, Philippe; Rosso, Marie-Noëlle

    2011-01-01

    Similarly to microbial pathogens, plant-parasitic nematodes secrete into their host plants proteins that are essential to establish a functional interaction. Identifying the destination of nematode secreted proteins within plant cell compartment(s) will provide compelling clues on their molecular functions. Here the fine localization of five nematode secreted proteins was analysed throughout parasitism in Arabidopsis thaliana. An immunocytochemical method was developed that preserves both the host and the pathogen tissues, allowing the localization of nematode secreted proteins within both organisms. One secreted protein from the amphids and three secreted proteins from the subventral oesophageal glands involved in protein degradation and cell wall modification were secreted in the apoplasm during intercellular migration and to a lower extent by early sedentary stages during giant cell formation. Conversely, another protein produced by both subventral and dorsal oesophageal glands in parasitic stages accumulated profusely at the cell wall of young and mature giant cells. In addition, secretion of cell wall-modifying proteins by the vulva of adult females suggested a role in egg laying. The study shows that the plant apoplasm acts as an important destination compartment for proteins secreted during migration and during sedentary stages of the nematode. PMID:21115667

  15. Antigenic secreted proteins from Haemophilus paragallinarum. A 110-kDa putative RTX protein.

    PubMed

    Mena-Rojas, Erika; Vázquez Cruz, Candelario; Vaca Pacheco, Sergio; García González, Octavio; Pérez-Márquez, Víctor M; Pérez-Méndez, Alma; Ibarra-Caballero, Jorge; de la Garza, Mireya; Zenteno, Edgar; Negrete-Abascal, Erasmo

    2004-03-12

    Haemophilus paragallinarum is the causal agent of infectious coryza, an economically important disease for the poultry industry. This bacterium secreted proteins of 25-110 kDa during its growth in brain heart infusion, tryptic soy broth, or Luria-Bertani glucose phosphate media, all lacking serum. Some of these proteins were recognized by sera from chickens experimentally infected with H. paragallinarum. A 110-kDa protein was recognized by a serum pool from convalescent-phase pigs naturally infected with Actinobacillus pleuropneumoniae, and also by a rabbit polyclonal serum against Apx I as well as a rabbit serum against Mannheimia haemolytica leukotoxin, suggesting the presence of an RTX-like protein in H. paragallinarum. H. paragallinarum secreted proteins could be important immunogens in the control of infectious coryza.

  16. The Secrets of Acinetobacter Secretion.

    PubMed

    Weber, Brent S; Kinsella, Rachel L; Harding, Christian M; Feldman, Mario F

    2017-02-16

    Infections caused by the bacterial pathogen Acinetobacter baumannii are a mounting concern for healthcare practitioners as widespread antibiotic resistance continues to limit therapeutic treatment options. The biological processes used by A. baumannii to cause disease are not well defined, but recent research has indicated that secreted proteins may play a major role. A variety of mechanisms have now been shown to contribute to protein secretion by A. baumannii and other pathogenic species of Acinetobacter, including a type II secretion system (T2SS), a type VI secretion system (T6SS), autotransporter, and outer membrane vesicles (OMVs). In this review, we summarize the current knowledge of secretion systems in Acinetobacter species, and highlight their unique aspects that contribute to the pathogenicity and persistence of these emerging pathogens.

  17. Protein secretion in Pseudomonas aeruginosa: the xcpA gene encodes an integral inner membrane protein homologous to Klebsiella pneumoniae secretion function protein PulO.

    PubMed Central

    Bally, M; Ball, G; Badere, A; Lazdunski, A

    1991-01-01

    xcp mutations have pleiotropic effects on the secretion of proteins in Pseudomonas aeruginosa PAO. The nucleotide sequence of a 1.2-kb DNA fragment that complements the xcp-1 mutation has been determined. Sequence analysis shows the xcpA gene product to be a 31.8-kDa polypeptide, with a highly hydrophobic character. This is consistent with a localization in the cytoplasmic membrane in P. aeruginosa, determined after specific expression of the xcpA gene under control of the T7 phi 10 promoter. A very strong homology was found between XcpA and PulO, a membrane protein required for pullulanase secretion in Klebsiella pneumoniae. This suggests the existence of a signal sequence-dependent secretion process common to these two unrelated gram-negative bacteria. Images PMID:1898929

  18. A novel two-component system involved in secretion stress response in Streptomyces lividans.

    PubMed

    Gullón, Sonia; Vicente, Rebeca L; Mellado, Rafael P

    2012-01-01

    Misfolded proteins accumulating outside the bacterial cytoplasmic membrane can interfere with the secretory machinery, hence the existence of quality factors to eliminate these misfolded proteins is of capital importance in bacteria that are efficient producers of secretory proteins. These bacteria normally use a specific two-component system to respond to the stress produced by the accumulation of the misfolded proteins, by activating the expression of HtrA-like proteases to specifically eliminate the incorrectly folded proteins. Overproduction of alpha-amylase in S. lividans causing secretion stress permitted the identification of a two-component system (SCO4156-SCO4155) that regulates three HtrA-like proteases which appear to be involved in secretion stress response. Mutants in each of the genes forming part of the two-genes operon that encodes the sensor and regulator protein components accumulated misfolded proteins outside the cell, strongly suggesting the involvement of this two-component system in the S. lividans secretion stress response. To our knowledge this is the first time that a specific secretion stress response two-component system is found to control the expression of three HtrA-like protease genes in S. lividans, a bacterium that has been repeatedly used as a host for the synthesis of homologous and heterologous secretory proteins of industrial application.

  19. Therapeutic Potential of Secreted Amyloid Precursor Protein APPsα

    PubMed Central

    Mockett, Bruce G.; Richter, Max; Abraham, Wickliffe C.; Müller, Ulrike C.

    2017-01-01

    Cleavage of the amyloid precursor protein (APP) by α-secretase generates an extracellularly released fragment termed secreted APP-alpha (APPsα). Not only is this process of interest due to the cleavage of APP within the amyloid-beta sequence, but APPsα itself has many physiological properties that suggest its great potential as a therapeutic target. For example, APPsα is neurotrophic, neuroprotective, neurogenic, a stimulator of protein synthesis and gene expression, and enhances long-term potentiation (LTP) and memory. While most early studies have been conducted in vitro, effectiveness in animal models is now being confirmed. These studies have revealed that either upregulating α-secretase activity, acutely administering APPsα or chronic delivery of APPsα via a gene therapy approach can effectively treat mouse models of Alzheimer’s disease (AD) and other disorders such as traumatic head injury. Together these findings suggest the need for intensifying research efforts to harness the therapeutic potential of this multifunctional protein. PMID:28223920

  20. Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana

    PubMed Central

    Karuppanan, Kalimuthu; Duhra-Gill, Sifti; Kailemia, Muchena J.; Phu, My L.; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Rodriguez, Raymond L.; Nandi, Somen; McDonald, Karen A.

    2017-01-01

    Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50%) N-glycan structure was GlcNAc2(Xyl)Man3(Fuc)GlcNAc2 in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting. PMID:28054967

  1. Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana.

    PubMed

    Karuppanan, Kalimuthu; Duhra-Gill, Sifti; Kailemia, Muchena J; Phu, My L; Lebrilla, Carlito B; Dandekar, Abhaya M; Rodriguez, Raymond L; Nandi, Somen; McDonald, Karen A

    2017-01-04

    Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50%) N-glycan structure was GlcNAc₂(Xyl)Man₃(Fuc)GlcNAc₂ in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting.

  2. Protein Secretion in Gram-Positive Bacteria: From Multiple Pathways to Biotechnology.

    PubMed

    Anné, Jozef; Economou, Anastassios; Bernaerts, Kristel

    2016-11-25

    A number of Gram-positive bacteria are important players in industry as producers of a diverse array of economically interesting metabolites and proteins. As discussed in this overview, several Gram-positive bacteria are valuable hosts for the production of heterologous proteins. In contrast to Gram-negative bacteria, proteins secreted by Gram-positive bacteria are released into the culture medium where conditions for correct folding are more appropriate, thus facilitating the isolation and purification of active proteins. Although seven different protein secretion pathways have been identified in Gram-positive bacteria, the majority of heterologous proteins are produced via the general secretion or Sec pathway. Not all proteins are equally well secreted, because heterologous protein production often faces bottlenecks including hampered secretion, susceptibility to proteases, secretion stress, and metabolic burden. These bottlenecks are associated with reduced yields leading to non-marketable products. In this chapter, besides a general overview of the different protein secretion pathways, possible hurdles that may hinder efficient protein secretion are described and attempts to improve yield are discussed including modification of components of the Sec pathway. Attention is also paid to omics-based approaches that may offer a more rational approach to optimize production of heterologous proteins.

  3. 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

  4. A translocator-specific export signal establishes the translocator-effector secretion hierarchy that is important for type III secretion system function.

    PubMed

    Tomalka, Amanda G; Stopford, Charles M; Lee, Pei-Chung; Rietsch, Arne

    2012-12-01

    Type III secretion systems are used by many Gram-negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host-cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore-forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the Pseudomonas aeruginosa translocator protein PopD as a model to identify its export signals. The N-terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone binding site and one at the very C-terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator-effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells. © 2012 Blackwell Publishing Ltd.

  5. A translocator-specific export signal establishes the translocator-effector secretion hierarchy that is important for type III secretion system function

    PubMed Central

    Tomalka, Amanda G.; Stopford, Charles M.; Lee, Pei-Chung; Rietsch, Arne

    2012-01-01

    Summary Type III secretion systems are used by many Gram-negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host-cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore-forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the P. aeruginosa translocator protein PopD as a model to identify its export signals. The amino-terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone-binding site and one at the very C-terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator-effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells. PMID:23121689

  6. Characterization of the secretome of chickpea suspension culture reveals pathway abundance and the expected and unexpected secreted proteins.

    PubMed

    Gupta, Sonika; Wardhan, Vijay; Verma, Shikha; Gayali, Saurabh; Rajamani, Uma; Datta, Asis; Chakraborty, Subhra; Chakraborty, Niranjan

    2011-11-04

    The secretome of an organism is defined as a set of secreted proteins that encompasses all proteins exported to the extracellular space. To better understand the chickpea secretome, we used callus culture to isolate and identify secreted proteins as a step toward determining their functions. Proteins in the extracellular media of the suspension culture were examined using SDS-PAGE and mass spectrometry (LC-MS/MS). Proteomic analysis led to the identification of 773 proteins, presumably involved in a variety of functions including metabolism, signal transduction, transport, and cell defense, in addition to maintaining redox status of extracellular space. Bioinformatic analysis confirmed 724 proteins, accounting for 94% of the identified proteins, as constituents of the secretome. Analysis of the secretome revealed the presence of several proteins of unknown function and a large number of classical and nonclassical secreted proteins. This represents the first comprehensive secretome of a legume genome, which is yet to be sequenced. Comparative analysis of the chickpea secretome with those of Medicago, Arabidopsis, and rice revealed that the majority of identified proteins are seemingly species-specific. This study demonstrates that characterization of the chickpea secretome in vitro can be used to identify secreted proteins, which has implications for systems biology research.

  7. Type III secretion as a generalizable strategy for the production of full-length biopolymer-forming proteins.

    PubMed

    Azam, Anum; Li, Cheng; Metcalf, Kevin J; Tullman-Ercek, Danielle

    2016-11-01

    Biopolymer-forming proteins are integral in the development of customizable biomaterials, but recombinant expression of these proteins is challenging. In particular, biopolymer-forming proteins have repetitive, glycine-rich domains and, like many heterologously expressed proteins, are prone to incomplete translation, aggregation, and proteolytic degradation in the production host. This necessitates tailored purification processes to isolate each full-length protein of interest from the truncated forms as well as other contaminating proteins; owing to the repetitive nature of these proteins, the truncated polypeptides can have very similar chemistry to the full-length form and are difficult to separate from the full-length protein. We hypothesized that bacterial expression and secretion would be a promising alternative option for biomaterials-forming proteins, simplifying isolation of the full-length target protein. By using a selective secretion system, truncated forms of the protein are not secreted and thus are not found in the culture harvest. We show that a synthetically upregulated type III secretion system leads to a general increase in secretion titer for each protein that we tested. Moreover, we observe a substantial enhancement in the homogeneity of full-length forms of pro-resilin, tropo-elastin crosslinking domains, and silk proteins produced in this manner, as compared with proteins purified from the cytosol. Secretion via the type III apparatus limits co-purification of truncated forms of the target protein and increases protein purity without extensive purification steps. Demonstrating the utility of such a system, we introduce several modifications to resilin-based peptides and use an un-optimized, single-column process to purify these proteins. The resulting materials are of sufficiently high quantity and yield for the production of antimicrobial hydrogels with highly reproducible rheological properties. The ease of this process and its

  8. Pachytene spermatocyte protein(s) stimulate Sertoli cells grown in bicameral chambers: dose-dependent secretion of ceruloplasmin, sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin.

    PubMed

    Onoda, M; Djakiew, D

    1991-03-01

    Interactions between pachytene spermatocytes and Sertoli cells were investigated using the bicameral culture chamber system. Pachytene spermatocytes were isolated from adult rats with a purity in excess of 90% by centrifugal elutriation. The pachytene spermatocytes were cultured in a defined media and pachytene spermatocyte protein prepared from the conditioned media by dialysis and lyophilization. This pachytene spermatocyte protein was reconstituted at various concentrations and incubated with confluent epithelial sheets of immature Sertoli cells cultured in bicameral chambers. Pachytene spermatocyte protein stimulated secretion of total [35S]methionine-labeled protein from Sertoli cells in a dose-dependent manner predominantly in an apical direction. This stimulatory effect of pachytene spermatocyte protein was domain specific from the apical surface of Sertoli cells, and seemed specific for secretion because total intracellular protein did not increase under the influence of pachytene spermatocyte protein. Pachytene spermatocyte protein and follicle-stimulating hormone additively stimulated Sertoli cell secretion. The physicochemical characteristics of the stimulatory pachytene spermatocyte protein are indicative of heat stability, whereas the stimulatory pachytene spermatocyte protein exhibit acid, dithiothreitol and trypsin sensitivity, and partial urea sensitivity. Furthermore, Sertoli cell secretion of ceruloplasmin, sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin in response to various concentrations of pachytene spermatocyte protein were determined by immunoprecipitate of these [35S]methionine-labeled proteins with polyclonal antibodies. Maximal stimulation of ceruloplasmin and sulfated glycoprotein-1 secretion from Sertoli cells was observed at a dose of 50 micrograms/ml pachytene spermatocyte protein, whereas maximal stimulation of sulfated glycoprotein-2 and transferrin secretion from Sertoli cells was observed at a dose of 100

  9. 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.

  10. The type VI secretion system: a multipurpose delivery system with a phage-like machinery.

    PubMed

    Records, Angela R

    2011-07-01

    Whether they live in the soil, drift in the ocean, survive in the lungs of human hosts or reside on the surfaces of leaves, all bacteria must cope with an array of environmental stressors. Bacteria have evolved an impressive suite of protein secretion systems that enable their survival in hostile environments and facilitate colonization of eukaryotic hosts. Collectively, gram-negative bacteria produce six distinct secretion systems that deliver proteins to the extracellular milieu or directly into the cytosol of host cells. The type VI secretion system (T6SS) was discovered recently and is encoded in at least one fourth of all sequenced gram-negative bacterial genomes. T6SS proteins are evolutionarily and structurally related to phage proteins, and it is likely that the T6SS apparatus is reminiscent of phage injection machinery. Most studies of T6SS function have been conducted in the context of host-pathogen interactions. However, the totality of data suggests that the T6SS is a versatile tool with roles in virulence, symbiosis, interbacterial interactions, and antipathogenesis. This review gives a brief history of T6SS discovery and an overview of the pathway's predicted structure and function. Special attention is paid to research addressing the T6SS of plant-associated bacteria, including pathogens, symbionts and plant growth-promoting rhizobacteria.

  11. 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

  12. 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

  13. Analysis of Exocyst-Positive Organelle (EXPO)-Mediated Unconventional Protein Secretion (UPS) in Plant Cells.

    PubMed

    Ding, Yu; Wang, Juan

    2017-01-01

    Unconventional protein secretion (UPS) together with conventional protein secretion (CPS) is responsible for protein secretion in plants. We have previously identified a novel UPS pathway in plants, which is mediated by exocyst-positive organelle-EXPO. Here, we describe detailed protocols to study UPS in plants by using Arabidopsis protoplasts or transgenic suspension cells, expressing the EXPO marker Exo70E2-XFP, as materials. Via drug and osmotic treatment plus secretion assay, we illustrate several major methods to analyze EXPO-mediated UPS in plant cells, which also supplys mining tools for similar study.

  14. 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.

  15. 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.

  16. Calcium-dependent activator protein for secretion 2 (CAPS2) promotes BDNF secretion and is critical for the development of GABAergic interneuron network.

    PubMed

    Shinoda, Yo; Sadakata, Tetsushi; Nakao, Kazuhito; Katoh-Semba, Ritsuko; Kinameri, Emi; Furuya, Asako; Yanagawa, Yuchio; Hirase, Hajime; Furuichi, Teiichi

    2011-01-04

    Calcium-dependent activator protein for secretion 2 (CAPS2) is a dense-core vesicle-associated protein that is involved in the secretion of BDNF. BDNF has a pivotal role in neuronal survival and development, including the development of inhibitory neurons and their circuits. However, how CAPS2 affects BDNF secretion and its biological significance in inhibitory neurons are largely unknown. Here we reveal the role of CAPS2 in the regulated secretion of BDNF and show the effect of CAPS2 on the development of hippocampal GABAergic systems. We show that CAPS2 is colocalized with BDNF, both synaptically and extrasynaptically in axons of hippocampal neurons. Overexpression of exogenous CAPS2 in hippocampal neurons of CAPS2-KO mice enhanced depolarization-induced BDNF exocytosis events in terms of kinetics, frequency, and amplitude. We also show that in the CAPS2-KO hippocampus, BDNF secretion is reduced, and GABAergic systems are impaired, including a decreased number of GABAergic neurons and their synapses, a decreased number of synaptic vesicles in inhibitory synapses, and a reduced frequency and amplitude of miniature inhibitory postsynaptic currents. Conversely, excitatory neurons in the CAPS2-KO hippocampus were largely unaffected with respect to field excitatory postsynaptic potentials, miniature excitatory postsynaptic currents, and synapse number and morphology. Moreover, CAPS2-KO mice exhibited several GABA system-associated deficits, including reduced late-phase long-term potentiation at CA3-CA1 synapses, decreased hippocampal theta oscillation frequency, and increased anxiety-like behavior. Collectively, these results suggest that CAPS2 promotes activity-dependent BDNF secretion during the postnatal period that is critical for the development of hippocampal GABAergic networks.

  17. Calcium-dependent activator protein for secretion 2 (CAPS2) promotes BDNF secretion and is critical for the development of GABAergic interneuron network

    PubMed Central

    Shinoda, Yo; Sadakata, Tetsushi; Nakao, Kazuhito; Katoh-Semba, Ritsuko; Kinameri, Emi; Furuya, Asako; Yanagawa, Yuchio; Hirase, Hajime; Furuichi, Teiichi

    2011-01-01

    Calcium-dependent activator protein for secretion 2 (CAPS2) is a dense-core vesicle-associated protein that is involved in the secretion of BDNF. BDNF has a pivotal role in neuronal survival and development, including the development of inhibitory neurons and their circuits. However, how CAPS2 affects BDNF secretion and its biological significance in inhibitory neurons are largely unknown. Here we reveal the role of CAPS2 in the regulated secretion of BDNF and show the effect of CAPS2 on the development of hippocampal GABAergic systems. We show that CAPS2 is colocalized with BDNF, both synaptically and extrasynaptically in axons of hippocampal neurons. Overexpression of exogenous CAPS2 in hippocampal neurons of CAPS2-KO mice enhanced depolarization-induced BDNF exocytosis events in terms of kinetics, frequency, and amplitude. We also show that in the CAPS2-KO hippocampus, BDNF secretion is reduced, and GABAergic systems are impaired, including a decreased number of GABAergic neurons and their synapses, a decreased number of synaptic vesicles in inhibitory synapses, and a reduced frequency and amplitude of miniature inhibitory postsynaptic currents. Conversely, excitatory neurons in the CAPS2-KO hippocampus were largely unaffected with respect to field excitatory postsynaptic potentials, miniature excitatory postsynaptic currents, and synapse number and morphology. Moreover, CAPS2-KO mice exhibited several GABA system-associated deficits, including reduced late-phase long-term potentiation at CA3–CA1 synapses, decreased hippocampal theta oscillation frequency, and increased anxiety-like behavior. Collectively, these results suggest that CAPS2 promotes activity-dependent BDNF secretion during the postnatal period that is critical for the development of hippocampal GABAergic networks. PMID:21173225

  18. 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

  19. Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

    PubMed Central

    Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

    2013-01-01

    The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

  20. 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,…

  1. Inside the Chamber of Secrets of the Type III Secretion System.

    PubMed

    Cascales, Eric

    2017-03-09

    The bacterial type III secretion system is a specialized machine that injects effectors into eukaryotic cells to manipulate the host cell physiology. In this issue of Cell, Hu et al. use cryo-electron tomography to reveal an unprecedented level of details regarding the architecture of this machine and the conformational changes that occur during its assembly.

  2. 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,…

  3. Yersinia pestis Yop secretion protein F: purification, characterization, and protective efficacy against bubonic plague.

    PubMed

    Swietnicki, Wieslaw; Powell, Bradford S; Goodin, Jeremy

    2005-07-01

    Yersinia pestis is a gram-negative human pathogen that uses a type III secretion system to deliver virulence factors into human hosts. The delivery is contact-dependent and it has been proposed that polymerization of Yop secretion protein F (YscF) is used to puncture mammalian cell membranes to facilitate delivery of Yersinia outer protein effectors into host cells. To evaluate the potential immunogenicity and protective efficacy of YscF against Y. pestis, we used a purified recombinant YscF protein as a potential vaccine candidate in a mouse subcutaneous infection model. YscF was expressed and purified from Escherichia coli by immobilized metal-ion affinity chromatography and protein identity was confirmed by ion trap mass spectrometry. The recombinant protein was highly alpha-helical and formed relatively stable aggregates under physiological conditions. The properties were consistent with behavior expected for the native YscF, suggesting that the antigen was properly folded. Ten mice were inoculated subcutaneously, administered booster injections after one month, and challenged with 130 LD(50) of wild type Y. pestis CO92. Six animals in the vaccinated group but none in the control group survived the challenge. The vaccinated animals produced high levels of specific antibodies against YscF as determined by Western blot. The data were statistically significant (P = 0.053 by two-tailed Fisher's test), suggesting that the YscF protein can provide a protective immune response against lethal plague challenge during subcutaneous plague infection.

  4. Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium.

    PubMed

    Knobloch, Denise; Ostermann, Kai; Rödel, Gerhard

    2012-01-01

    Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium, indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

  5. Role of autocleavage in the function of a type III secretion specificity switch protein in Salmonella enterica serovar Typhimurium.

    PubMed

    Monjarás Feria, Julia V; Lefebre, Matthew D; Stierhof, York-Dieter; Galán, Jorge E; Wagner, Samuel

    2015-10-13

    Type III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the "switch protein," a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of the Salmonella enterica serovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage event per se does not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function. Bacterial interaction with eukaryotic hosts often involves complex molecular machines for targeted delivery of bacterial effector proteins. One such machine, the type III secretion system of some Gram-negative bacteria, serves to inject a multitude of structurally

  6. 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

  7. 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.

  8. A novel strategy for protein production using non-classical secretion pathway in Bacillus subtilis.

    PubMed

    Chen, Jingqi; Zhao, Liuqun; Fu, Gang; Zhou, Wenjuan; Sun, Yuanxia; Zheng, Ping; Sun, Jibin; Zhang, Dawei

    2016-04-28

    The Gram-positive bacterium Bacillus subtilis has been widely used as a cell factory for the production of proteins due to its generally regarded as safe (GRAS) nature and secretion capability. Of the known secretory pathways in B. subtilis, the majority of proteins are exported from the cytoplasm by Sec pathway, Tat pathway and ABC transporters, etc. However, the production of heterologous proteins by B. subtilis is unfortunately not that straight forward because of the bottlenecks in classical secretion pathways. The aim of this work is to explore a new method for protein production based on non-classical secretion pathway. One D-psicose 3-epimerase (RDPE) which converts D-fructose into D-psicose from Ruminococcus sp. 5_1_39BFAA was successfully and substantially secreted into the extracellular milieu without the direction of signal peptide. Subsequently, we demonstrated that RDPE contained no native signal peptide, and the secretion of RDPE was not dependent on Sec or Tat pathway or due to cell lysis, which indicated that RDPE is a non-classically secreted protein. Then, we attempted to evaluate the possibility of using RDPE as a signal to export eighteen reporter proteins into the culture medium. Five of eleven homologous proteins, two of five heterologous proteins from other bacterium and two heterologous proteins of eukaryotic source were successfully secreted into the extracellular milieu at different secretion levels when they were fused to RDPE mediated by a flexible 21-bp linker to keep a distance between two single proteins. Furthermore, the secretion rates of two fusion proteins (RDPE-DnaK and RDPE-RFP) reached more than 50 %. In addition, most of the fusion proteins retained enzyme or biological activity of their corresponding target proteins, and all of the fusions still had the activity of RDPE. We found and identified a heterologous non-classically secreted protein RDPE, and showed that RDPE could direct proteins of various types into the culture

  9. The ATP-dependent ClpXP and Lon proteases regulate expression of the Yersinia pestis type III secretion system via regulated proteolysis of YmoA, a small histone-like protein.

    PubMed

    Jackson, Michael W; Silva-Herzog, Eugenia; Plano, Gregory V

    2004-12-01

    The Yersinia pestis plasmid pCD1-encoded type III secretion system (T3SS) is essential for the pathogenicity of Y. pestis in mammalian hosts. T3SS-associated genes are maximally expressed at 37 degrees C in the absence of extracellular calcium. Expression of T3SS genes requires LcrF, an AraC-like transcriptional activator, and is repressed by YmoA, a small histone-like protein. The mechanism by which temperature regulates T3SS gene expression has not been determined; however, changes in DNA topology have been implicated in this process. We report here that a Y. pestis strain deficient in production of the ClpXP and Lon proteases does not express a functional T3SS partly because of high cytosolic levels of YmoA. YmoA is rapidly degraded at 37 degrees C in wild-type Y. pestis, but remains stable in a clpXPlon deletion mutant. The stability of YmoA in wild-type Y. pestis increased as the growth temperature of the culture decreased; in contrast, YmoA was stable at all temperatures examined in the clpXPlon deletion mutant. These results indicate that the ClpXP and Lon proteases contribute to the environmental regulation of the Y. pestis T3SS system through regulated proteolysis of YmoA.

  10. Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: proteins secreted by Brevibacillus choshinensis.

    PubMed

    Tanio, Michikazu; Tanaka, Rikou; Tanaka, Takeshi; Kohno, Toshiyuki

    2009-03-15

    Here we report the first application of amino acid-type selective (AATS) isotope labeling of a recombinant protein secreted by Brevibacillus choshinensis for a nuclear magnetic resonance (NMR) study. To prepare the 15N-AATS-labeled protein, the transformed B. choshinensis was cultured in 15N-labeled amino acid-containing C.H.L. medium, which is commonly used in the Escherichia coli expression system. The analyses of the 1H-15N heteronuclear single quantum coherence (HSQC) spectra of the secreted proteins with a 15N-labeled amino acid demonstrated that alanine, arginine, asparagine, cysteine, glutamine, histidine, lysine, methionine, and valine are suitable for selective labeling, although acidic and aromatic amino acids are not suitable. The 15N labeling for glycine, isoleucine, leucine, serine, and threonine resulted in scrambling to specific amino acids. These results indicate that the B. choshinensis expression system is an alternative tool for AATS labeling of recombinant proteins, especially secretory proteins, for NMR analyses.

  11. Multidrug transporter ABCG2/breast cancer resistance protein secretes riboflavin (vitamin B2) into milk.

    PubMed

    van Herwaarden, Antonius E; Wagenaar, Els; Merino, Gracia; Jonker, Johan W; Rosing, Hilde; Beijnen, Jos H; Schinkel, Alfred H

    2007-02-01

    The multidrug transporter breast cancer resistance protein (BCRP/ABCG2) is strongly induced in the mammary gland during pregnancy and lactation. We here demonstrate that BCRP is responsible for pumping riboflavin (vitamin B(2)) into milk, thus supplying the young with this important nutrient. In Bcrp1(-/-) mice, milk secretion of riboflavin was reduced >60-fold compared to that in wild-type mice. Yet, under laboratory conditions, Bcrp1(-/-) pups showed no riboflavin deficiency due to concomitant milk secretion of its cofactor flavin adenine dinucleotide, which was not affected. Thus, two independent secretion mechanisms supply vitamin B(2) equivalents to milk. BCRP is the first active riboflavin efflux transporter identified in mammals and the first transporter shown to concentrate a vitamin into milk. BCRP activity elsewhere in the body protects against xenotoxins by reducing their absorption and mediating their excretion. Indeed, Bcrp1 activity increased excretion of riboflavin into the intestine and decreased its systemic availability in adult mice. Surprisingly, the paradoxical dual utilization of BCRP as a xenotoxin and a riboflavin pump is evolutionarily conserved among mammals as diverse as mice and humans. This study establishes the principle that an ABC transporter can transport a vitamin into milk and raises the possibility that other vitamins and nutrients are likewise secreted into milk by ABC transporters.

  12. Multidrug Transporter ABCG2/Breast Cancer Resistance Protein Secretes Riboflavin (Vitamin B2) into Milk▿

    PubMed Central

    van Herwaarden, Antonius E.; Wagenaar, Els; Merino, Gracia; Jonker, Johan W.; Rosing, Hilde; Beijnen, Jos H.; Schinkel, Alfred H.

    2007-01-01

    The multidrug transporter breast cancer resistance protein (BCRP/ABCG2) is strongly induced in the mammary gland during pregnancy and lactation. We here demonstrate that BCRP is responsible for pumping riboflavin (vitamin B2) into milk, thus supplying the young with this important nutrient. In Bcrp1−/− mice, milk secretion of riboflavin was reduced >60-fold compared to that in wild-type mice. Yet, under laboratory conditions, Bcrp1−/− pups showed no riboflavin deficiency due to concomitant milk secretion of its cofactor flavin adenine dinucleotide, which was not affected. Thus, two independent secretion mechanisms supply vitamin B2 equivalents to milk. BCRP is the first active riboflavin efflux transporter identified in mammals and the first transporter shown to concentrate a vitamin into milk. BCRP activity elsewhere in the body protects against xenotoxins by reducing their absorption and mediating their excretion. Indeed, Bcrp1 activity increased excretion of riboflavin into the intestine and decreased its systemic availability in adult mice. Surprisingly, the paradoxical dual utilization of BCRP as a xenotoxin and a riboflavin pump is evolutionarily conserved among mammals as diverse as mice and humans. This study establishes the principle that an ABC transporter can transport a vitamin into milk and raises the possibility that other vitamins and nutrients are likewise secreted into milk by ABC transporters. PMID:17145775

  13. Size and conformation limits to secretion of disulfide-bonded loops in autotransporter proteins.

    PubMed

    Leyton, Denisse L; Sevastsyanovich, Yanina R; Browning, Douglas F; Rossiter, Amanda E; Wells, Timothy J; Fitzpatrick, Rebecca E; Overduin, Michael; Cunningham, Adam F; Henderson, Ian R

    2011-12-09

    Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the secretion of autotransporters requires a translocation-competent conformation for the passenger domain that differs markedly from the structure of the fully folded secreted protein. The nature of the translocation-competent conformation remains controversial, in particular whether the passenger domain can adopt secondary structural motifs, such as disulfide-bonded segments, while maintaining a secretion-competent state. Here, we used the endogenous and closely spaced cysteine residues of the plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli to investigate the effect of disulfide bond-induced folding on translocation of an autotransporter passenger domain. We reveal that rigid structural elements within disulfide-bonded segments are resistant to autotransporter-mediated secretion. We define the size limit of disulfide-bonded segments tolerated by the autotransporter system demonstrating that, when present, cysteine pairs are intrinsically closely spaced to prevent congestion of the translocator pore by large disulfide-bonded regions. These latter data strongly support the hairpin mode of autotransporter biogenesis.

  14. 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.

  15. 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

  16. Entamoeba histolytica infection and secreted proteins proteolytically damage enteric neurons.

    PubMed

    Lourenssen, Sandra; Houpt, Eric R; Chadee, Kris; Blennerhassett, Michael G

    2010-12-01

    The enteric protozoan parasite Entamoeba histolytica causes amebic colitis through disruption of the mucus layer, followed by binding to and destruction of epithelial cells. However, it is not known whether ameba infections or ameba components can directly affect the enteric nervous system. Analysis of mucosal innervations in the mouse model of cecal amebiasis showed that axon density was diminished to less than 25% of control. To determine whether amebas directly contributed to axon loss, we tested the effect of either E. histolytica secreted products (Eh-SEC) or soluble components (Eh-SOL) to an established coculture model of myenteric neurons, glia, and smooth muscle cells. Neuronal survival and axonal degeneration were measured after 48 h of exposure to graded doses of Eh-SEC or Eh-SOL (10 to 80 μg/ml). The addition of 80 μg of either component/ml decreased the neuron number by 30%, whereas the axon number was decreased by 50%. Cytotoxicity was specific to the neuronal population, since the glial and smooth muscle cell number remained similar to that of the control, and was completely abrogated by prior heat denaturation. Neuronal damage was partially prevented by the cysteine protease inhibitor E-64, showing that a heat-labile protease was involved. E. histolytica lysates derived from amebas deficient in the major secreted protease EhCP5 caused a neurotoxicity similar to that of wild-type amebas. We conclude that E. histolytica infection and ameba protease activity can cause selective damage to enteric neurons.

  17. 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

  18. A novel plant cell bioproduction platform for high-yield secretion of recombinant proteins.

    PubMed

    Xu, Jianfeng; Kieliszewski, Marcia J

    2012-01-01

    Plant cell suspension culture integrates the merits of whole-plant systems with those of microbial fermentation and mammalian cell culture, and has been recognized as a promising alternative biosynthetic platform for valuable proteins. However, the low protein productivity dilemma has been the bottleneck toward commercializing this technology. Here, we describe a new technology, termed hydroxyproline (Hyp)-Glyco technology, that dramatically increases the yield of secreted recombinant proteins from cultured plant cells by expressing them as fusions with a novel glycomodule tag comprising an Hyp-rich repetitive peptide (HypRP) backbone that is subsequently glycosylated through the Hyp residues. The extensive glycosylation of the HypRP tags greatly extends the serum half-life of small therapeutic proteins, such as interferon α2b or human growth hormone, without significantly impairing their bioactivities and the tag greatly enhances solubility.

  19. 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

    ... Prospective Grant of Exclusive License: Secreted Frizzled Related Protein-1 (sFRP-1) and derivatives thereof... 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 and uses thereof....

  20. Harpins, multifunctional proteins secreted by gram-negative plant-pathogenic bacteria.

    PubMed

    Choi, Min-Seon; Kim, Wooki; Lee, Chanhui; Oh, Chang-Sik

    2013-10-01

    Harpins are glycine-rich and heat-stable proteins that are secreted through type III secretion system in gram-negative plant-pathogenic bacteria. Many studies show that these proteins are mostly targeted to the extracellular space of plant tissues, unlike bacterial effector proteins that act inside the plant cells. Over the two decades since the first harpin of pathogen origin, HrpN of Erwinia amylovora, was reported in 1992 as a cell-free elicitor of hypersensitive response (HR), diverse functional aspects of harpins have been determined. Some harpins were shown to have virulence activity, probably because of their involvement in the translocation of effector proteins into plant cytoplasm. Based on this function, harpins are now considered to be translocators. Their abilities of pore formation in the artificial membrane, binding to lipid components, and oligomerization are consistent with this idea. When harpins are applied to plants directly or expressed in plant cells, these proteins trigger diverse beneficial responses such as induction of defense responses against diverse pathogens and insects and enhancement of plant growth. Therefore, in this review, we will summarize the functions of harpins as virulence factors (or translocators) of bacterial pathogens, elicitors of HR and immune responses, and plant growth enhancers.

  1. Evolution of conjugation and type IV secretion systems.

    PubMed

    Guglielmini, Julien; de la Cruz, Fernando; Rocha, Eduardo P C

    2013-02-01

    Genetic exchange by conjugation is responsible for the spread of resistance, virulence, and social traits among prokaryotes. Recent works unraveled the functioning of the underlying type IV secretion systems (T4SS) and its distribution and recruitment for other biological processes (exaptation), notably pathogenesis. We analyzed the phylogeny of key conjugation proteins to infer the evolutionary history of conjugation and T4SS. We show that single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) conjugation, while both based on a key AAA(+) ATPase, diverged before the last common ancestor of bacteria. The two key ATPases of ssDNA conjugation are monophyletic, having diverged at an early stage from dsDNA translocases. Our data suggest that ssDNA conjugation arose first in diderm bacteria, possibly Proteobacteria, and then spread to other bacterial phyla, including bacterial monoderms and Archaea. Identifiable T4SS fall within the eight monophyletic groups, determined by both taxonomy and structure of the cell envelope. Transfer to monoderms might have occurred only once, but followed diverse adaptive paths. Remarkably, some Firmicutes developed a new conjugation system based on an atypical relaxase and an ATPase derived from a dsDNA translocase. The observed evolutionary rates and patterns of presence/absence of specific T4SS proteins show that conjugation systems are often and independently exapted for other functions. This work brings a natural basis for the classification of all kinds of conjugative systems, thus tackling a problem that is growing as fast as genomic databases. Our analysis provides the first global picture of the evolution of conjugation and shows how a self-transferrable complex multiprotein system has adapted to different taxa and often been recruited by the host. As conjugation systems became specific to certain clades and cell envelopes, they may have biased the rate and direction of gene transfer by conjugation within prokaryotes.

  2. Antibacterial Flavonoids from Medicinal Plants Covalently Inactivate Type III Protein Secretion Substrates.

    PubMed

    Tsou, Lun K; Lara-Tejero, María; RoseFigura, Jordan; Zhang, Zhenrun J; Wang, Yen-Chih; Yount, Jacob S; Lefebre, Matthew; Dossa, Paul D; Kato, Junya; Guan, Fulan; Lam, Wing; Cheng, Yung-Chi; Galán, Jorge E; Hang, Howard C

    2016-02-24

    Traditional Chinese Medicines (TCMs) have been historically used to treat bacterial infections. However, the molecules responsible for these anti-infective properties and their potential mechanisms of action have remained elusive. Using a high-throughput assay for type III protein secretion in Salmonella enterica serovar Typhimurium, we discovered that several TCMs can attenuate this key virulence pathway without affecting bacterial growth. Among the active TCMs, we discovered that baicalein, a specific flavonoid from Scutellaria baicalensis, targets S. Typhimurium pathogenicity island-1 (SPI-1) type III secretion system (T3SS) effectors and translocases to inhibit bacterial invasion of epithelial cells. Structurally related flavonoids present in other TCMs, such as quercetin, also inactivated the SPI-1 T3SS and attenuated S. Typhimurium invasion. Our results demonstrate that specific plant metabolites from TCMs can directly interfere with key bacterial virulence pathways and reveal a previously unappreciated mechanism of action for anti-infective medicinal plants.

  3. 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 Central

    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

    2016-01-01

    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

  4. 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.

  5. The Ca(2+)-dependent activator protein for secretion CAPS: do I dock or do I prime?

    PubMed

    Stevens, David R; Rettig, Jens

    2009-02-01

    The "Ca(2+)-dependent activator protein for secretion" (CAPS) is a protein which reconstitutes regulated secretion in permeabilized neuroendocrine cells. It is generally accepted that CAPS plays an important role in the release of the contents of dense core vesicles in the nervous system as well as in a variety of other secretory tissues. At which step in the exocytotic process CAPS functions as well as its role in the fusion of synaptic vesicles is still under dispute. A recent growth spurt in the CAPS field has been fueled by genetic approaches in Caenorhabditis elegans and Drosophila as well as the application of knockout and knockdown approaches in mouse cells and in cell lines, respectively. We have attempted to review the body of work that established CAPS as an important regulator of secretion and to describe new information that has furthered our understanding of how CAPS may function. We discuss the conclusions, point out areas where controversy remains, and suggest directions for future experiments.

  6. Gibberellic acid-induced aleurone layers responding to heat shock or tunicamycin provide insight into the N-glycoproteome, protein secretion, and endoplasmic reticulum stress.

    PubMed

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

    2014-02-01

    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.

  7. 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

  8. Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization.

    PubMed

    Heiss, Silvia; Puxbaum, Verena; Gruber, Clemens; Altmann, Friedrich; Mattanovich, Diethard; Gasser, Brigitte

    2015-07-01

    Secretion leaders are required to direct nascent proteins to the secretory pathway. They are of interest in the study of intracellular protein transport, and are required for the production of secretory recombinant proteins. Secretion leaders are processed in two steps in the endoplasmic reticulum and Golgi. Although yeast cells typically contain about 150 proteins entering the secretory pathway, only a low number of proteins are actually secreted to the cell supernatant. Analysis of the secretome of the yeast Pichia pastoris revealed that the most abundant secretory protein, which we named Epx1, belongs to the cysteine-rich secretory protein family CRISP. Surprisingly, the Epx1 secretion leader undergoes a three-step processing on its way to the cell exterior instead of the usual two-step processing. The Kex2 cleavage site within the P. pastoris Epx1 leader is not conserved in the homologues of most other yeasts. We studied the effect of exchanging the Kex2-cleavage motif on the secretory behaviour of reporter proteins fused to variants of the Epx1 leader sequence, and observed mistargeting for some but not all of the variants using fluorescence microscopy. By targeting several recombinant human proteins for secretion, we revealed that a short variant of the leader sequence, as well as the Epx1 signal sequence alone, resulted in the correct N-termini of the secreted proteins. Both leader variants proved to be very efficient, even exceeding the secretion levels obtained with commonly used secretion leaders. Taken together, the novel Epx1 secretion leader sequences are a valuable tool for recombinant protein production as well as basic research of intracellular transport.

  9. 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

  10. Host-Pathogen Interactions: VII. Plant Pathogens Secrete Proteins which Inhibit Enzymes of the Host Capable of Attacking the Pathogen.

    PubMed

    Albersheim, P; Valent, B S

    1974-05-01

    The results presented demonstrate that microbial pathogens of plants have the ability to secrete proteins which effectively inhibit an enzyme synthesized by the host; an enzyme whose substrate is a constituent of the cell wall of the pathogen. The system in which this was discovered is the anthracnose-causing fungal pathogen (Colletotrichum lindemuthianum) and its host, the French bean (Phaseolus vulgaris). An endo-beta-1, 3-glucanase present in the bean leaves is specifically inhibited by a protein secreted by C. lindemuthianum. The cell walls of C. lindemuthianum are shown to be composed largely of a 1, 3-glucan.

  11. 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

  12. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  16. Integrated single-cell analysis shows Pichia pastoris secretes protein stochastically.

    PubMed

    Love, Kerry Routenberg; Panagiotou, Vasiliki; Jiang, Bo; Stadheim, Terrance A; Love, J Christopher

    2010-06-01

    The production of heterologous proteins by secretion from cellular hosts is an important determinant for the cost of biotherapeutics. A single-cell analytical method called microengraving was used to examine the heterogeneity in secretion by the methylotrophic yeast Pichia pastoris. We show that constitutive secretion of a human Fc fragment by P. pastoris is not cell-cycle dependent, but rather fluctuates between states of high and low productivity in a stochastic manner.

  17. 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

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

    USDA-ARS?s Scientific Manuscript database

    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, ...

  19. Autoproteolysis of YscU of Yersinia pseudotuberculosis is important for regulation of expression and secretion of Yop proteins.

    PubMed

    Björnfot, Ann-Catrin; Lavander, Moa; Forsberg, Ake; Wolf-Watz, Hans

    2009-07-01

    YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU(CC). Autoproteolysis occurs at the conserved N downward arrowPTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca(2+) conditions) and calcium-depleted medium (-Ca(2+) conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca(2+)-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca(2+) conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under -Ca(2+) conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.

  20. Biophysical characterization of the type III secretion tip proteins and the tip proteins attached to bacterium-like particles.

    PubMed

    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

    2015-02-01

    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 (PA) domain. In this study, the tip proteins IpaD, SipD, and LcrV belonging to type III 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.

  1. 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

  2. Synthesis and secretion of proteins by released malarial parasites.

    PubMed

    Elmendorf, H G; Bangs, J D; Haldar, K

    1992-06-01

    Controlled mechanical homogenization of Plasmodium falciparum-infected erythrocytes releases parasites of a quality sufficient for studying the export of newly synthesized plasmodial proteins. Protein synthesis occurs within intact released parasites as defined by resistance of acid-insoluble incorporation of radiolabel to high levels of exogenously added EDTA, hexokinase, and RNaseA. While exogenously added ATP and erythrocyte cytosol were not essential for biosynthetic activity at levels comparable to that seen in infected erythrocytes, the addition of an extracellular ATP regenerating system (ARS) stimulated the synthesis of parasite proteins. Conversely, parasite viability and biosynthetic activity are decreased by the addition of a non-hydrolyzable ATP analogue (ATP gamma S), ADP, or ATP in the absence of a regenerating system. These data suggest a metabolic interdependence between extracellular energy metabolism and biosynthetic functions within the parasite. The export of a predominant subset of proteins was retarded in the presence of Brefeldin A, indicating the existence of a classical secretory pathway characteristic of that seen in higher eukaryotic cells. Interestingly, a Brefeldin A-insensitive component of export was also consistently observed; this may suggest the existence of an additional alternative secretory mechanism in malaria.

  3. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. 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

  5. The Endoplasmic Reticulum Coat Protein II Transport Machinery Coordinates Cellular Lipid Secretion and Cholesterol Biosynthesis*

    PubMed Central

    Fryer, Lee G. D.; Jones, Bethan; Duncan, Emma J.; Hutchison, Claire E.; Ozkan, Tozen; Williams, Paul A.; Alder, Olivia; Nieuwdorp, Max; Townley, Anna K.; Mensenkamp, Arjen R.; Stephens, David J.; Dallinga-Thie, Geesje M.; Shoulders, Carol C.

    2014-01-01

    Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions. PMID:24338480

  6. 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.

  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. 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

  9. 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.

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

    PubMed Central

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

    2013-01-01

    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. The outer membrane vesicles: Secretion system type zero.

    PubMed

    Guerrero-Mandujano, Andrea; Hernández-Cortez, Cecilia; Ibarra, Jose Antonio; Castro-Escarpulli, Graciela

    2017-07-01

    Gram-negative bacteria have mechanisms through which they can colonize and survive in different environments, such as the secretion systems types (1-6) that have been widely studied and characterized. Nowadays, some authors have proposed extracellular structures, such as the outer membrane vesicles (OMVs), to be considered as an additional and independent secretion system. The OMVs are spherical particles of 50-250 nm in diameter; they originate in the outer membrane, and therefore they have a very similar composition to the latter. These particles can transport an important variety of biomolecules: enzymes, toxins, antigenic determinants and even nucleic acids. Thus, it is of great interest to collect data describing the advantages of the transport of biomolecules through the OMVs and, thus, determine their role as a potential secretion system. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. LMAN1 (ERGIC-53) is a potential carrier protein for matrix metalloproteinase-9 glycoprotein secretion

    PubMed Central

    Duellman, Tyler; Burnett, John; Shin, Alice; Yang, Jay

    2015-01-01

    Matrix metalloproteinase-9 (MMP-9) is a secreted glycoprotein with a major role in shaping the extra-cellular matrix and a detailed understanding of the secretory mechanism could help identify methods to correct diseases resulting from dysregulation of secretion. MMP-9 appears to follow a canonical secretory pathway through a quality control cycle in the endoplasmic reticulum (ER) before transport of the properly folded protein to the Golgi apparatus and beyond for secretion. Through a complementation assay, we determined that LMAN1, a well-studied lectin-carrier protein, interacts with a secretion-competent N-glycosylated MMP-9 in the ER while N-glycosylation-deficient secretion-compromised MMP-9 does not. In contrast, co-immunoprecipitation demonstrated protein interaction between LMAN1 and secretion-compromised N-glycosylation-deficient MMP-9. MMP-9 secretion was reduced in the LMAN1 knockout cell line compared to control cells confirming the functional role of LMAN1. These observations support the role of LMAN1 as a lectin-carrier protein mediating efficient MMP-9 secretion. PMID:26150355

  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. LMAN1 (ERGIC-53) is a potential carrier protein for matrix metalloproteinase-9 glycoprotein secretion.

    PubMed

    Duellman, Tyler; Burnett, John; Shin, Alice; Yang, Jay

    2015-08-28

    Matrix metalloproteinase-9 (MMP-9) is a secreted glycoprotein with a major role in shaping the extracellular matrix and a detailed understanding of the secretory mechanism could help identify methods to correct diseases resulting from dysregulation of secretion. MMP-9 appears to follow a canonical secretory pathway through a quality control cycle in the endoplasmic reticulum (ER) before transport of the properly folded protein to the Golgi apparatus and beyond for secretion. Through a complementation assay, we determined that LMAN1, a well-studied lectin-carrier protein, interacts with a secretion-competent N-glycosylated MMP-9 in the ER while N-glycosylation-deficient secretion-compromised MMP-9 does not. In contrast, co-immunoprecipitation demonstrated protein interaction between LMAN1 and secretion-compromised N-glycosylation-deficient MMP-9. MMP-9 secretion was reduced in the LMAN1 knockout cell line compared to control cells confirming the functional role of LMAN1. These observations support the role of LMAN1 as a lectin-carrier protein mediating efficient MMP-9 secretion.

  15. Breast cancer resistance protein (Bcrp1/Abcg2) reduces systemic exposure of the dietary carcinogens aflatoxin B1, IQ and Trp-P-1 but also mediates their secretion into breast milk.

    PubMed

    van Herwaarden, Antonius E; Wagenaar, Els; Karnekamp, Barbara; Merino, Gracia; Jonker, Johan W; Schinkel, Alfred H

    2006-01-01

    The breast cancer resistance protein (BCRP/ABCG2) usually protects the body from a wide variety of environmental and dietary xenotoxins by reducing their net uptake from intestine and by increasing their hepatobiliary, intestinal and renal elimination. BCRP is also highly expressed in lactating mammary glands in mice, and this expression is conserved in cows and humans. As a result, BCRP substrates can be secreted into milk. We investigated whether different classes of dietary carcinogens are substrates of Bcrp1/BCRP and the implications for systemic exposure and breast milk contamination. Using polarized cell lines, we found that Bcrp1 transports the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and the potent human hepatocarcinogen aflatoxin B1, and decreases their cellular accumulation up to 10-fold. In vivo pharmacokinetic studies showed that [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin B1 plasma levels were substantially lower in wild-type compared with Bcrp1-/- mice, after both oral and intravenous administration, demonstrating that Bcrp1 restricts systemic exposure to these carcinogens. Moreover, Bcrp1 mediates transfer of [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin into milk, with 3.4+/-0.6, 2.6+/-0.3 and 3.8+/-0.5-fold higher milk to plasma ratios, respectively, in lactating wild-type versus Bcrp1-/- mice. We have thus identified Bcrp1/BCRP as one of the molecular mechanisms by which heterocyclic amines and aflatoxin are transferred into milk, thereby posing a health risk to breast-fed infants and dairy consumers. Paradoxically, Bcrp1/BCRP appears to have both protective and adverse roles with respect to exposure to dietary carcinogens.

  16. 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

  17. Effective Information Systems: What's the Secret?

    ERIC Educational Resources Information Center

    Kirkham, Sandi

    1994-01-01

    Argues that false assumptions about user needs implicit in methodologies for building information systems have resulted in inadequate and inflexible systems. Checkland's Soft Systems Methodology is examined as a useful alternative. Its fundamental features are described, and examples of models demonstrate how the methodology can facilitate…

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

    PubMed Central

    Shindo, Takayuki; Kaschani, Farnusch; 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.

    2016-01-01

    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. LcrQ and SycH function together at the Ysc type III secretion system in Yersinia pestis to impose a hierarchy of secretion.

    PubMed

    Wulff-Strobel, Christine R; Williams, Andrew W; Straley, Susan C

    2002-01-01

    LcrQ is a regulatory protein unique to Yersinia. Previous study in Yersinia pseudotuberculosis and Yersinia enterocolitica prompted the model in which LcrQ negatively regulates the expression of a set of virulence proteins called Yops, and its secretion upon activation of the Yop secretion (Ysc) type III secretion system permits full induction of Yops expression. In this study, we tested the hypothesis that LcrQ's effects on Yops expression might be indirect. Excess LcrQ was found to exert an inhibitory effect specifically at the level of Yops secretion, independent of production, and a normal inner Ysc gate protein LcrG was required for this activity. However, overexpression of LcrQ did not prevent YopH secretion, suggesting that LcrQ's effects at the Ysc discriminate among the Yops. We tested this idea by determining the effects of deletion or overexpression of LcrQ, YopH and their common chaperone SycH on early Yop secretion through the Ysc. Together, our findings indicated that LcrQ is not a negative regulator directly, but it acts in partnership with SycH at the Ysc gate to control the entry of a set of Ysc secretion substrates. A hierarchy of YopH secretion before YopE appears to be imposed by SycH in conjunction with both LcrQ and YopH. LcrQ and SycH in addition influenced the deployment of LcrV, a component of the Yops delivery mechanism. Accordingly, LcrQ appears to be a central player in determining the substrate specificity of the Ysc.

  20. Regulation of the Yersinia type III secretion system: traffic control

    PubMed Central

    Dewoody, Rebecca S.; Merritt, Peter M.; Marketon, Melanie M.

    2013-01-01

    Yersinia species, as well as many other Gram-negative pathogens, use a type III secretion system (T3SS) to translocate effector proteins from the bacterial cytoplasm to the host cytosol. This T3SS resembles a molecular syringe, with a needle-like shaft connected to a basal body structure, which spans the inner and outer bacterial membranes. The basal body of the injectisome shares a high degree of homology with the bacterial flagellum. Extending from the T3SS basal body is the needle, which is a polymer of a single protein, YscF. The distal end of the needle serves as a platform for the assembly of a tip complex composed of LcrV. Though never directly observed, prevailing models assume that LcrV assists in the insertion of the pore-forming proteins YopB and YopD into the host cell membrane. This completes a bridge between the bacterium and host cell to provide a continuous channel through which effectors are delivered. Significant effort has gone into understanding how the T3SS is assembled, how its substrates are recognized and how substrate delivery is controlled. Arguably the latter topic is the least understood; however, recent advances have provided new insight, and therefore, this review will focus primarily on summarizing the current state of knowledge regarding the control of substrate delivery by the T3SS. Specifically, we will discuss the roles of YopK, as well as YopN and YopE, which have long been linked to regulation of translocation. We also propose models whereby the YopK regulator communicates with the basal body of the T3SS to control translocation. PMID:23390616

  1. Secreted Frizzled-Related Protein 2 and Inflammation-Induced Skeletal Muscle Atrophy.

    PubMed

    Zhu, Xiaoxi; Kny, Melanie; Schmidt, Franziska; Hahn, Alexander; Wollersheim, Tobias; Kleber, Christian; Weber-Carstens, Steffen; Fielitz, Jens

    2017-02-01

    In sepsis, the disease course of critically ill patients is often complicated by muscle failure leading to ICU-acquired weakness. The myokine transforming growth factor-β1 increases during inflammation and mediates muscle atrophy in vivo. We observed that the transforming growth factor-β1 inhibitor, secreted frizzled-related protein 2, was down-regulated in skeletal muscle of ICU-acquired weakness patients. We hypothesized that secreted frizzled-related protein 2 reduction enhances transforming growth factor-β1-mediated effects and investigated the interrelationship between transforming growth factor-β1 and secreted frizzled-related protein 2 in inflammation-induced atrophy. Observational study and prospective animal trial. Two ICUs and research laboratory. Twenty-six critically ill patients with Sequential Organ Failure Assessment scores greater than or equal to 8 underwent a skeletal muscle biopsy from the vastus lateralis at median day 5 in ICU. Four patients undergoing elective orthopedic surgery served as controls. To search for signaling pathways enriched in muscle of ICU-acquired weakness patients, a gene set enrichment analysis of our recently published gene expression profiles was performed. Quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry were used to analyze secreted frizzled-related protein 2 expression and protein content. A mouse model of inflammation-induced skeletal muscle atrophy due to polymicrobial sepsis and cultured myocytes were used for mechanistic analyses. None. Gene set enrichment analysis uncovered transforming growth factor-β1 signaling activation in vastus lateralis from ICU-acquired weakness patients. Muscular secreted frizzled-related protein 2 expression was reduced after 5 days in ICU. Likewise, muscular secreted frizzled-related protein 2 expression was decreased early and continuously in mice with inflammation-induced atrophy. In muscle, secreted frizzled-related protein 2

  2. Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery.

    PubMed

    Panavas, Tadas; Lu, Jin; Liu, Xuesong; Winkis, Ann-Marie; Powers, Gordon; Naso, Michael F; Amegadzie, Bernard

    2011-09-01

    Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity.

  3. 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.

  4. Disulphide bridge formation of proinsulin fusion proteins during secretion in Streptomyces.

    PubMed

    Vértesy, L; Tripier, D; Koller, K P; Riess, G

    1991-03-01

    To study disulphide bridge formation by Streptomyces lividans TK 24 in secreted single chain precursors of insulin a fusion protein (PTF 1) was investigated consisting of monkey proinsulin and the aminoterminal sequence Asp1 to Gly43 of the alpha-amylase inhibitor tendamistat from Streptomyces tendae. The purified soluble protein PTF 1 has a molecular mass of 14.4 kDa. The primary structure was elucidated after digestion with lysyl endopeptidase and fragment analysis. In this system, disulphide bond formation occurs in a way that the first cysteine in proinsulin is linked to the next following cysteine in the amino-acid chain resulting in a non-natural folding of the insulin part of the fusion protein. Re-folding of PTF 1 by reduction and