Academic literature on the topic 'Type II secretion system ; T2SS'
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Journal articles on the topic "Type II secretion system ; T2SS"
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Michel, Gérard P. F., Anthony Aguzzi, Geneviève Ball, Chantal Soscia, Sophie Bleves, and Romé Voulhoux. "Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium." Microbiology 157, no. 7 (July 1, 2011): 1945–54. http://dx.doi.org/10.1099/mic.0.045849-0.
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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.
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Douzi, Badreddine, Alain Filloux, and Romé Voulhoux. "On the path to uncover the bacterial type II secretion system." Philosophical Transactions of the Royal Society B: Biological Sciences 367, no. 1592 (April 19, 2012): 1059–72. http://dx.doi.org/10.1098/rstb.2011.0204.
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Gram-negative bacteria have evolved several secretory pathways to release enzymes or toxins into the surrounding environment or into the target cells. The type II secretion system (T2SS) is conserved in Gram-negative bacteria and involves a set of 12 to 16 different proteins. Components of the T2SS are located in both the inner and outer membranes where they assemble into a supramolecular complex spanning the bacterial envelope, also called the secreton. The T2SS substrates transiently go through the periplasm before they are translocated across the outer membrane and exposed to the extracellular milieu. The T2SS is unique in its ability to promote secretion of large and sometimes multimeric proteins that are folded in the periplasm. The present review describes recently identified protein–protein interactions together with structural and functional advances in the field that have contributed to improve our understanding on how the type II secretion apparatus assembles and on the role played by individual proteins of this highly sophisticated system.
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Strozen, Timothy G., Gang Li, and S. Peter Howard. "YghG (GspSβ) Is a Novel Pilot Protein Required for Localization of the GspSβType II Secretion System Secretin of Enterotoxigenic Escherichia coli." Infection and Immunity 80, no. 8 (May 14, 2012): 2608–22. http://dx.doi.org/10.1128/iai.06394-11.
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ABSTRACTThe enterotoxigenicEscherichia coli(ETEC) pathotype, characterized by the prototypical strain H10407, is a leading cause of morbidity and mortality in the developing world. A major virulence factor of ETEC is the type II secretion system (T2SS) responsible for secretion of the diarrheagenic heat-labile enterotoxin (LT). In this study, we have characterized the two type II secretion systems, designated alpha (T2SSα) and beta (T2SSβ), encoded in the H10407 genome and describe the prevalence of both systems in otherE. colipathotypes. Under laboratory conditions, the T2SSβis assembled and functional in the secretion of LT into culture supernatant, whereas the T2SSαis not. Insertional inactivation of the three genes located upstream ofgspCβ(yghJ,pppA, andyghG) in the atypical T2SSβoperon revealed that YghJ is not required for assembly of the GspDβsecretin or secretion of LT, that PppA is likely the prepilin peptidase required for the function of T2SSβ, and that YghG is required for assembly of the GspDβsecretin and thus function of the T2SSβ. Mutational and physiological analysis further demonstrated that YghG (redesignated GspSβ) is a novel outer membrane pilotin protein that is integral for assembly of the T2SSβby localizing GspDβto the outer membrane, whereupon GspDβforms the macromolecular secretin multimer through which T2SSβsubstrates are translocated.
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Michel, Gérard P. F., Eric Durand, and Alain Filloux. "XphA/XqhA, a Novel GspCD Subunit for Type II Secretion in Pseudomonas aeruginosa." Journal of Bacteriology 189, no. 10 (March 9, 2007): 3776–83. http://dx.doi.org/10.1128/jb.00205-07.
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ABSTRACT The opportunistic human pathogen bacterium Pseudomonas aeruginosa secretes various exoproteins in its surrounding environment. Protein secretion involves different secretory systems, including the type II secretion system, or T2SS, that is one of the most efficient secretory pathways of P. aeruginosa. There are two T2SS in this bacterium, the quorum-sensing-regulated Xcp system and the Hxc system, which is only present under phosphate-limiting conditions. Like T2SS of other bacteria, the Xcp T2SS is species specific, and this specificity mainly involves two proteins, XcpP (GspC family) and the secretin XcpQ (GspD family), which are the gatekeepers of the system. Interestingly, an orphan secretin, XqhA, was previously reported as being able to functionally replace the XcpQ secretin. In this study, we identified another gene, which we named xphA (xcpP homologue A), which is located next to xqhA. We showed that deletion of the xphA gene in an xcpP mutant caused the disappearance of the residual secretion observed in this mutant strain, indicating that the protein XphA plays a role in the secretion process. Our results also revealed that complementation of an xcpP/xcpQ mutant can be obtained with the gene couple xphA/xqhA. The XphA and XqhA proteins (the PAQA subunit) could thus form, together with XcpR-Z, a functional hybrid T2SS. A two-dimensional polyacrylamide gel electrophoresis analysis showed that except for the aminopeptidase PaAP, for which secretion is not restored by the PAQA subunit in the xcpP/xcpQ deletion mutant, each major Xcp-dependent exoprotein is secreted by the new hybrid machinery. Our work supports the idea that components of the GspC/GspD families, such as XphA/XqhA or XcpP/XcpQ, are assembled as a specific tandem within the T2SS. Each of these pairs may thus confer a different level of secretion specificity, as is the case with respect to PaAP. Finally, using a chromosomal xphA-lacZ fusion, we showed that the xphA-xqhA genes are transcribed from an early stage of bacterial growth. We thus suggest that the PAQA subunit might be involved in the secretion process at a different growth stage than XcpP/XcpQ.
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Burtnick, Mary N., Paul J. Brett, and David DeShazer. "Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM." Infection and Immunity 82, no. 8 (May 27, 2014): 3214–26. http://dx.doi.org/10.1128/iai.01739-14.
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ABSTRACTBurkholderia pseudomallei, the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provideB. pseudomalleiwith a growth advantagein vitroandin vivo, but relatively little is known about the full repertoire of exoproducts associated with each system. In this study, we constructed deletion mutations ingspDandgspE, T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles ofB. pseudomalleiMSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants ofB. pseudomalleiMSHR668 andB. pseudomalleiΔgspDgrown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of ΔgspDstrains. Many of these proteins were putative hydrolytic enzymes, including 12 proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities is T2SS dependent. Previous studies had failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of theB. pseudomalleiT2SS secretome.
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Lu, Connie, Young-un Park, Konstantin Korotkov, Wei Mi, Stewart Turley, Veer Bhatt, Ripal Shah, and Wim Hol. "Multiple approaches towards understanding the type II secretion system." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C577. http://dx.doi.org/10.1107/s2053273314094224.
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Transport of folded proteins across membranes is a feat accomplished by few biomacromolecular machines. One of the machineries able to do so is the sophisticated type II secretion system (T2SS). It can translocate key virulence factors from the bacterial periplasm into the lumen of the gut of the human host. A prime example is the secretion of cholera toxin by Vibrio cholerae. The T2SS consists of ~12 different proteins, most of these present in multiple copies, organized into three subassemblies: (i) the Inner Membrane Platform; (ii) the Pseudopilus in the periplasm, which acts most likely as a piston pushing exoproteins through the outer membrane pore; (iii) the Outer Membrane Complex, allowing passage of ~100 kDa folded proteins. We have determined crystal structures from more than a dozen T2SS domains, yet, a full understanding of the architecture and mechanism of action of the T2SS remains a formidable challenge. Our approaches include the use of "assistant-multimers" to promote recalcitrant multimer formation and of nanobodies to overcome reluctant crystal formation. The Inner Membrane Platform is interacting with the secretion ATPase GspE which most likely needs to be hexameric for full activity. Full-length GspE co-crystallized with its major partner, the cytoplasmic domain of GspL, revealed a tremendous flexibility of this ATPase, and, most unexpectedly, also the organization of the same linear arrangement of cyto-GspL domains throughout three entirely different crystal forms. Two very different hexamers of GspE were elucidated by linking the GspE subunit to the subunit of Hcp1, which successfully acted as an "assistant hexamer", inducing hexamer formation by GspE. The dodecameric nature of the ~ 850 kDa GspD, the major component of the Outer Membrane Complex, evident in earlier electron microscopy studies, was observed in the dodecameric ring-like helix in crystals of its N-terminal domain. The contacts between GspD and the inner-membrane protein GspC will be discussed as well as the remarkably frequent occurrence of dimers of Inner Membrane Platform domains. How dimers are co-assembled with an ATPase hexamer with C6 symmetry and the Outer Membrane Complex dodecamer with C12 symmetry remains one of the many fascinating outstanding questions of the T2SS.
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Maltz, Michele, and Joerg Graf. "The Type II Secretion System Is Essential for Erythrocyte Lysis and Gut Colonization by the Leech Digestive Tract SymbiontAeromonas veronii." Applied and Environmental Microbiology 77, no. 2 (November 19, 2010): 597–603. http://dx.doi.org/10.1128/aem.01621-10.
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ABSTRACTHemolysin and the type II secretion system (T2SS) have been shown to be important for virulence in many pathogens, but very few studies have shown their importance in beneficial microbes. Here, we investigated the importance of the type II secretion pathway in the beneficial digestive-tract association ofAeromonas veroniiand the medicinal leechHirudo verbanaand revealed a critical role for the hemolysis of erythrocytes. A mutant with a miniTn5insertion inexeM, which is involved in forming the inner membrane platform in the T2SS, was isolated by screening mutants for loss of hemolysis on blood agar plates. A hemolysis assay was used to quantify the mutant's deficiency in lysing sheep erythrocytes and revealed a 99.9% decrease compared to the parent strain. The importance of the T2SS in the colonization of the symbiotic host was assessed. Colonization assays revealed that the T2SS is critical for initial colonization of the leech gut. The defect was tied to the loss of hemolysin production by performing a colonization assay with blood containing lysed erythrocytes. This restored the colonization defect in the mutant. Complementation of the mutant using the promoter region andexeMNrevealed that the T2SS is responsible for secreting hemolysin into the extracellular space and that both the T2SS and hemolysin export by the T2SS are critical for initial establishment ofA. veroniiin the leech gut.
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Zhang, Yichen, Frederick Faucher, and Zongchao Jia. "Insights into minor pseudopilin complexes of the Type II secretion system." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C585. http://dx.doi.org/10.1107/s2053273314094145.
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The type II secretion system (T2SS) is sophisticated multiprotein machinery that enables Gram-negative pathogens to secrete a wide range of exoproteins, named virulence factors, into the extracellular environments. In Pseudomonas aeruginosa, the Xcp T2SS is responsible for secreting many virulence factors that induce severe infections. In T2SS, the recognition and binding of secreted exoproteins are conducted by a structure called the pseudopilius tip, which is formed by four minor pseudopilins, including XcpU, XcpV, XcpW and XcpX. These minor pseudopilins form a quaternary complex, which is also involved in the initiation and regulation of the pseudopilus assembly. Although individual structures of these four pseudopilins have been revealed in different organisms, the substrate recognition and binding mechanisms have not been clearly elucidated due to the lack of systematic studies on the whole structures of several complexes formed by these pseudopilins. As a result, the understanding of the structures of these protein complexes will provide useful information for unveiling the mystery of the recognition and binding mechanisms. The establishment of the substrate binding model requires the preparation of stable complex(es) of substrates and certain minor pseudopilin(s). In this work, we aim to gradually elucidate the secretion mechanisms by assembling each component to build up the whole architecture. The structure of XcpV in complex with XcpW has been determined, and other complexes, especially the XcpU-containing binary and ternary complexes, have been stably established and purified. The identification of these complex structures will significantly promote our understandings of the type II secretion mechanisms.
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Baldi, Deborah L., Ellen E. Higginson, Dianna M. Hocking, Judyta Praszkier, Rosalia Cavaliere, Catherine E. James, Vicki Bennett-Wood, et al. "The Type II Secretion System and Its Ubiquitous Lipoprotein Substrate, SslE, Are Required for Biofilm Formation and Virulence of Enteropathogenic Escherichia coli." Infection and Immunity 80, no. 6 (March 26, 2012): 2042–52. http://dx.doi.org/10.1128/iai.06160-11.
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ABSTRACTEnteropathogenicEscherichia coli(EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenicE. coli. The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease.
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Shi, Liang, Shuang Deng, Matthew J. Marshall, Zheming Wang, David W. Kennedy, Alice C. Dohnalkova, Heather M. Mottaz, et al. "Direct Involvement of Type II Secretion System in Extracellular Translocation of Shewanella oneidensis Outer Membrane Cytochromes MtrC and OmcA." Journal of Bacteriology 190, no. 15 (May 23, 2008): 5512–16. http://dx.doi.org/10.1128/jb.00514-08.
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ABSTRACT MtrC and OmcA are cell surface-exposed lipoproteins important for reducing solid metal oxides. Deletions of type II secretion system (T2SS) genes reduced their extracellular release and their accessibility to the proteinase K treatment, demonstrating the direct involvement of T2SS in translocation of MtrC and OmcA to the bacterial cell surface.
Dissertations / Theses on the topic "Type II secretion system ; T2SS"
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Zhang, Hui. "Structural studies of the inner-membrane platform of the bacterial type II secretion system." Thesis, Queen Mary, University of London, 2018. http://qmro.qmul.ac.uk/xmlui/handle/123456789/42808.
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The type II secretion system (T2SS) is widespread in Gram-negative bacteria that cause disease in animals and plants. In human and animal pathogens toxins are secreted (e.g. cholera toxin) and in plant pathogens lytic enzymes that breakdown the plant cell wall are exported in to the extracellular milieu (e.g. pectate lyase). Structurally the T2SS comprises at least 11 core proteins that form three major subassemblies spanning the inner-membrane, periplasmic space and outer-membrane: (i) the inner-membrane platform and associated cytoplasmic ATPase (E); (ii) the pseudopilus, which consists of five pseudopilins, G to K; and (iii) a large, pore-forming outer-membrane complex secretin D. The inner-membrane platform comprises three single transmembrane helix proteins, and one three transmembrane helix protein, OutF. The evidence from cryo-electron microscopy on the related type IVa pilus machine (T4PS) places the protein corresponding to OutF at the centre of this platform. This platform is responsible for assembling the pilus and for communicating between the periplasm and the cytoplasmic ATPase. To date, no high-resolution structure of a full-length OutF/PilC family protein is available. A low-resolution electron microscopy reconstruction of isolated PilG (PilC ortholog from Neisseria meningitides T4PS) showed a tetrameric two lobed structure. Here I report the results of studying the structure of the inner-membrane protein OutF from Dickeya dadantii and the complete inner-membrane platform comprising 9 proteins: OutEFGHIJKLM. This work involved cloning the corresponding operon, purifying the proteins, and using crystallography and electron microscopy. Key results reported here are the crystal structure of the first cytoplasmic domain of Dickeya dadantii, OutF65-172 and a preliminary three-dimensional model of the Dickeya dadantii inner-membrane platform. This model, and higher-resolution models to come, will provide valuable information about the oligomeric state, and arrangement of the inner-membrane proteins. These studies will help us to understand how the type II secretion system works.
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Trinh, Thi Trang Nhung. "Structural studies of type IX and type II secretion systems." Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0089.
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Les protéines synthétisées et sécrétées par les bactéries jouent des rôles importants pour leur survie. Les bactéries à Gram négatif ont développé des voies de sécrétion en tant qu'armes principales pour transporter des facteurs de virulence dans l'environnement extracellulaire ou dans des cellules hôte. L'un de ces systèmes, le T9SS a été principalement étudié chez l'agent pathogène oral Porphyromonas gingivalis et chez la bactérie mobile Flavobacterium johnsoniae. Un autre complexe, le T2SS est le principal déterminant de la virulence de la bactérie Pseudomonas aeruginosa, un agent pathogène de la fibrose kystique. Dans le cadre de ma thèse, j'ai résolu la structure atomique de plusieurs composants centraux du T9SS et du T2SS. Concernant le projet T9SS, j'ai essayé de cristalliser le domaine cytoplasmique de GldL de F. johnsoniae. La co-cristallisation de GldL avec des Nbs a été réalisée sans succès. Néanmoins, les structures cristallines de deux nanobody contre GldL ont été résolues par remplacement moléculaire. De plus, j'ai également travaillé sur la protéine PG1058 de P. gingivalis. J'ai résolu sa structure par diffraction anomale à la longueur d’onde du selenium. Concernant le projet T2SS, je me suis concentré sur la partie N-terminale de XcpQ, une sous-unité de la sécrétine. J'ai résolu la structure cristalline de XcpQN012 seul et en complexe avec le nanobody vhh04 à une résolution de 2,98 Å et de 2,9 Å, respectivement. Enfin, j'ai participé à la détermination structurale de TssK, un composant de plaque de base du système de T6SS et déterminer la structure cristalline d'un nanobody contre le domaine périplasmique de PorMProteins synthesized and secreted by bacteria serve many important roles in their survival. In particular, Gram-negative bacteria have evolved secretion pathways as the main weapons for transporting virulence factors into target cells or into the extracellular environment. One of these systems, the type IX secretion system (T9SS) or the Por secretion system, has been studied mainly in the oral pathogen Porphyromonas gingivalis and the gliding bacterium Flavobacterium johnsoniae. Another complex, the type II secretion system (T2SS) is the main determinant of the virulence of Pseudomonas aeruginosa, a cystic fibrosis pathogen. In my PhD thesis, I solved the atomic structure of several core components of both T9SS and T2SS.For the T9SS project, I tried to crystallize the cytoplasmic domain of GldL from F. johnsoniae. The co-crystallization of GldL with Nbs was unsuccessfull. The crystal structures of two nanobodies against GldL were solved by molecular replacement. I also worked on the PG1058 protein of P. gingivalis. I obtained crystals of the selenomethionine-derivatized PG1058 OmpA_C-like domain that diffracted up to 1.55 Å, and solved its structure by single-wavelength anomalous diffraction. For the T2SS project, I focused on the N-terminal part of XcpQ, a subunit of the secretin. I solved the crystal structure of XcpQN012 alone and in complex with nanobody vhh04 at a resolution of 2.98 Å and 2.9 Å, respectively. In addition, I also took part in the structural determination of the base plate component TssK of the T6SS and determined the crystal structure of one nanobody (vhh19) against the periplasmic domain of PorM
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Gu, Shuang. "Secretin interactions in the type II secretion system." Thesis, Queen Mary, University of London, 2012. http://qmro.qmul.ac.uk/xmlui/handle/123456789/2482.
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The type II secretion system (T2SS) is the major terminal branch of the general secretory pathway. It is composed of 12-15 proteins, most in multiple copies, and spans the inner and outer membranes of Gram-negative bacteria. The T2SS secretin subunits form a large dodecameric torus-like structure in the outer membrane. The secretin is the only essential component in the outer membrane and secreted proteins and virulence factors pass through the pore in the toroidal secretin dodecamer and out into the environment. The interaction between the secretin and its partners plays a key role in regulation of the T2SS. The interaction between the so-called homology region of the innermembrane protein GspC (GspC-HR) and secretin provides the structural and functional integrity of the secretion machinery across the two cell membranes. The interaction between secretin and its pilotin translocates the secretin subunits to the outer membrane. In this Thesis, the interactions between secretin and its partners are studied at molecular level. The GspC-HR structure is solved using NMR spectroscopy. Its interaction with secretin (GspD) is elucidated using several biochemical and biophysical approaches and a model of the complex is proposed. Also, the interaction between secretin (GspD) and pilotin (GspS) is further charicterisied. An 18 residues secretin sequence is identified as responsible for interacting with pilotin. Upon binding to the pilotin, the unstructured secretin forms a helical structure.
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Santos, Moreno Javier. "Molecular mechanism of pseudopilus assembly in the Klebsiella oxytoca type II secretion system." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC216/document.
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Le système de sécrétion de type II (SST2) permet la sécrétion de protéines repliées à travers la membrane externe chez les bactéries à Gram-négatif. Le SST2 est une nano-machine enchâssée dans l’enveloppe bactérienne, proche par sa composition et structure aux systèmes d’assemblage des pili de type IV (PT4) impliqués, entre autres, dans d’adhésion et motilité. Chez Klebsiella oxytoca, la surexpression des gènes pul codant le SST2 permet l’assemblage de pili composées des sous-unités PulG. Ceci suggère qu’en conditions physiologiques l’assemblage d’un pseudopilus périplasmique permet la sécrétion du substrat spécifique du SST2, la pullulanase. Dans ce projet nous avons exploré le mécanisme moléculaire de l’assemblage du pseudopilus en se focalisant sur les interactions de PulG avec les composants du SST2 dans la membrane interne. En utilisant l’approche de double-hybride bactérien, nous avons établi le réseau d’interactions de PulG avec les pseudopilins mineures PulH, I, J et K et avec la plateforme d’assemblage (PA). Pour valider ces interactions, nous avons combiné des techniques de biochimie (co-purification par affinité, pontage cystéine et chimique) avec des analyses fonctionnelles de sécrétion et de formation du pseudopilus. Nous avons mis en évidence des interactions entre PulG et les protéines de la PA, PulF et PulM, et nous avons analysé en détail l’interface PulG-PulM. Les résultats suggèrent la formation d’un complexe PulK-I-J-H-G dans la membrane interne impliqué dans des étapes précoces de la formation du pseudopilus, à travers les interactions de PulG et PulH avec PulM et PulF. Nos données expérimentales suggèrent un rôle majeur de PulM dans la sécrétion, vraisemblablement durant l’assemblage du SST2 et l’élongation du pseudopilus. Nos travaux collaboratifs mettant en jeu l'analyse par spectroscopie de masse et en dynamique moléculaire in silico révèlent le rôle essentiel des résidus conservés Glu5 et Thr2 de PulG, requis pour l’interaction avec PulM. Ces données suggèrent que Glu5 participe à l'extraction de PulG de la membrane, en neutralisant la charge positive de son peptide N-terminal par des interactions intramoleculaires. Ces résultats permettent d'établir un modèle détaillant les étapes initiales de l’assemblage des pseudopili dans la membrane interne, relevant pour de futures études sur le SST2 et nanomachines homologues. sécrétion de protéinespili de type 4 assemblage de fibres complexes protéiques membranairesinteractions protéine-protéinemicroscopie à immuno-fluorescence simulations en dynamique moléculairedouble-hybride bactérien spectrométrie de masse nanomachines bacteriennesThe type II secretion system (T2SS) drives the translocation of folded, periplasmic proteins across the outer membrane in Gram-negative bacteria. Secretion is carried out by an envelope-spanning nanomachine that is similar to the apparatus that builds type IV pili (T4P), bacterial surface filaments involved in adhesion, motility and other functions. In the Pul T2SS of Klebsiella oxytoca, overexpression of pul genes in plate-grown bacteria allows the assembly of T4P-like surface fibres made of PulG subunits, suggesting that a periplasmic pseudopilus fibre plays a role in the secretion of the type II substrate pullulanase under physiological conditions. In this project, we explored the molecular mechanism of pseudopilus assembly by focusing on the interaction between PulG and the T2SS inner membrane and pseudopili components. The network of interactions of PulG with the minor pseudopilins PulH, I, J and K and the assembly platform (AP) components was established using bacterial two-hybrid analysis. To validate these interactions, we combined biochemical approaches (affinity co-purification, chemical or cysteine cross-linking) with functional assays of secretion and pseudopilus formation. We provide evidence of the interaction between PulG and the AP proteins PulF and PulM, and delve into the PulG-PulM interface. Our results point to the formation of a PulK-I-J-H-G complex in the plasma membrane involved in early steps of fibre assembly, with a determinant role for PulG and PulH interaction with PulM and PulF. We obtained experimental evidence supporting a major role for PulM in pseudopilus assembly and protein secretion, probably by intervening in the assembly of the T2SS apparatus and in pseudopilus elongation. The results of experimental and in silico studies in collaboration with experts in mass spectrometry and molecular dynamics support the essential role of the highly conserved PulG residues Glu5 and Thr2, which participate in PulM binding. In addition, Glu5 probably favours PulG membrane extraction by neutralising its N-terminal positive charge through intra-molecular interaction. These findings shed new light on early membrane events during fibre assembly, and open new and exciting avenues in research on T2SSs and related nanomachines.protein secretiontype 4 pilifibre assemblymembrane protein complexprotein-protein interactionsimmunofluorescence microscopymolecular dynamics simulationsbacterial two-hybrid assaymass spectrometrybacterial nanomachines
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Singh, Sunny Kumar. "Biophysical characterisation of LcrH, a class II chaperone of the type III secretion system." Thesis, University of Sussex, 2015. http://sro.sussex.ac.uk/id/eprint/58058/.
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The type three-secretion system (T3SS) is a large and complex protein nano-machine that many gram-negative pathogens employ to infect host cells. A key structure of this machine is a proteinaceous pore that inserts into the target membrane and forms a channel for bacterial toxins to flow from bacteria into the host cell. The pore is mainly formed from two large membrane proteins called “translocators”. Importantly, effective secretion and thus pore formation of the translocators depends on their binding to and being transported by small specialized chaperones after synthesis in the bacterial cytosol. Recent crystal structures have shown these chaperones are formed from modular tetratricopeptide repeats (TPRs). However, each crystal structure produced different homodimeric structures, suggesting flexibility in their topology that may be of importance to function. Given the crucial role of the translocator chaperones, we investigated the conformational stability of the chaperone LcrH (Yersinia pestis). Mutational analysis coupled with analytical ultra-centrifugation and equilibrium chemical denaturations showed that LcrH is a weak and thermodynamically unstable dimer (KD ≈ 15 μM, ΔGH2O = 7.4 kcalmol-1). The modular TPR structure of the dimer allows it to readily unfold in a non-cooperative manner to a one-third unfolded dimeric intermediate (ΔGH2O = 1.7 kcalmol-1), before cooperatively unfolding to a monomeric denatured state (ΔGH2O = 5.7 kcalmol-1). Thus under physiological conditions the chaperone is able to populate C-terminally unravelled partially folded states, whilst being held together by its dimeric interface. Such ability suggests a “fly-casting” mechanism as a route to binding their far larger translocator cargo.
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Douzi, Badreddine. "La machinerie de sécrétion de type II Xcp de Pseudomonas aeruginosa : relations structure-fonction et interactome." Thesis, Aix-Marseille 1, 2011. http://www.theses.fr/2011AIX10086.
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Les bactéries à Gram négatif sont entourées par une enveloppe cellulaire qui, contrairement aux bactéries à Gram positif, possèdent une organisation membranaire complexe composée d’une membrane interne appelée généralement membrane cytoplasmique, un espace périplasmique contenant une matrice de peptidoglycane et une membrane externe asymétrique constituée d’une monocouche de phospholipides surmontée d’une assise de lipopolysaccharide (LPS). Afin de franchir cette barrière, les bactéries à Gram négatif ont développé différentes voies de sécrétions spécifiques dédiées à l’export des protéines (effecteurs) du milieu intracellulaire vers le milieu extracellulaire. Jusqu'à présent, six systèmes de sécrétion ont été identifiés chez ces bactéries. Chez Pseudomonas aeruginosa, une bactérie pathogène opportuniste, le système de sécrétion de type II appelé aussi sécréton Xcp constitue l’un des facteurs principales de sa virulence. Le sécréton Xcp est un complexe macromoléculaire formé par 12 protéines, nommées XcpAO et XcpPC-XcpZM. Ce complexe macromoléculaire est organisé en trois sous-complexes : i) une plateforme d’assemblage ancrée dans la membrane interne formé par les protéines XcpRESFYLZM ii) un pore de sécrétion localisé dans la membrane externe formé par l’oligomérisation d’une protéine appelé la sécrétine XcpQD. Le pore de sécrétion est connecté à la plateforme de la membrane interne par une protéine appelée XcpPC iii) un pseudopilus périplasmique sous forme de fibre hélicoïdale qui est formé par la multimérisation d’une protéine appelée la pseudopiline majeure XcpTG. D’autres protéines appelées les pseudopilines mineures XcpUH-VI-WJ-XK intègrent le pseudopilus. La première partie du travail effectué au cours de cette thèse a eu pour but d’étudier et de comprendre par des approches structurales, biochimiques et biophysiques le mécanisme d’assemblage des pseudopilines en pseudopilus. La deuxième partie de ce travail a porté sur l’étude des réseaux d’interactions entre les substrats sécrétés et les composants de la machinerie Xcp. Durant cette thèse, nous avons ainsi i) identifier grâce à l’étude des interactions protéine-protéine l’existence d’un complexe quaternaire entre les pseudopilines mineures XcpUH-VI-WJ-XK localisées au sommet du pseudopilus ii) déterminer les structures de la pseudopiline majeure XcpTG par RMN et de la pseudopiline mineure XcpWJ par cristallographie aux rayons X iii) déterminer les différents éléments du sécréton qui interagissent avec les exoprotéines du sécréton. Ce réseau d’interaction nous a permis de proposer un modèle de fonctionnement du sécréton qui élucide le cheminement des exoprotéines dans le sécréton afin qu’elles soient exportées vers le milieu extracellulaireGram-negative bacteria are characterized by a complex organization of their cell envelope composed by the inner membrane (IM) called cytoplasmic membrane, the periplasmic space containing a peptidoglycan layer and the outer membrane (OM) covered by the lipopolysaccharide matrix. Gram-negative bacteria have evolved several specialized machines called secretion systems to export their effectors from the intracellular medium to the extracellular milieu or to the host cells. Up to now, at least six secretion systems have been identified. In the opportunistic pathogen Pseudomonas aeruginosa, the type II secretion system called the Xcp secreton is the major pathway for the release of virulence factors. The Xcp secreton is a macromolecular complex composed by 12 proteins called XcpAO, XcpPC-XcpZM. This machinery is organized in 3 sub-complexes: i) the assembly platform localized in the IM implicating XcpRESFYLZM proteins ii) the OM pore composed by the oligomerization of the secretin XcpQD. The connection between the assembly platform and the secretin is performed by XcpPC anchored in the IM iii) a periplasmic pseudopilus consisting of the multimerization of the so-called major pseudopilin XcpTG. The pseudopilus is a helicoidally filament spanning the periplasmic area and pushing the substrate into the secretin pore. Four other proteins, the minor pseudopilins XcpUH-VI-WJ-XK, were found in the pseudopilus. In the present work we first focused on the study of the pseudopilus components by biochemical, biophysical and structural strategies to understand their assembly. Secondly, we investigate the protein interactome between periplasmic secreton component and secreted substrates. Thus, we revealed the presence of a quaternary complex composed by XcpUH-VI-WJ-XK located at the tip of the pseudopilus. To understand at atomic scale the regulation of the pseudopilus, we determined the structure of two components of the pseudopilus XcpTG by NMR and XcpWJ by X-ray crystallography. Using systematic protein-protein interaction studies between secreton components and purified exoproteins of Pseudomonas aeruginosa, we identified 5 proteins of the secreton able to interact with exoproteins. This interaction network allowed us to propose a model for the secretion process including the sequential steps followed by exoproteins inside the secreton to leave the cell envelop
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Salomonsson, Emelie. "The role of the Type IV pili system in the virulence of Francisella tularensis." Doctoral thesis, Umeå universitet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1656.
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Francisella tularensis is a Gram-negative intracellular pathogen causing the zoonotic disease tularemia. F. tularensis can be found almost all over the world and has been recovered from several animal species, even though the natural reservoir of the bacterium and parts of its life cycle are still unknown. Humans usually get infected after handling infected animals or from bites of blood-feeding arthropod vectors. There are four subspecies of F. tularensis: the highly virulent tularensis (Type A) that causes a very aggressive form of the disease, with mortality as high as 60% if untreated, the moderately virulent holarctica (Type B) and mediasiatica, and the essentially avirulent subspecies F. novicida. So far, our knowledge of the molecular mechanisms that would explain these differences in virulence among the subspecies is poor. However, recent developments of genetic tools and access to genomic sequences have laid the ground for progress in this research field. Analysis of genome sequences have identified several regions that differ between F. tularensis subspecies. One of these regions, RD19, encodes proteins postulated to be involved in assembly of type IV pili (Tfp), organelles that have been implicated in processes like twitching motility, biofilm formation and cell-to-cell communication in pathogenic bacteria. While there have been reports of pili-like structures on the surface of F. tularensis, these have not been linked to the Tfp encoding gene clusters until now. Herein, I present evidence that the Francisella pilin, PilA, can complement pilin-like characteristics and promote assembly of fibers in a heterologous system in Neisseria gonorrhoeae. pilA was demonstrated to be required for full virulence of both type A and type B strains in mice when infected via peripheral routes. A second region, RD18, encoding a protein unique to F. tularensis and without any known function, was verified to be essential for virulence in a type A strain. Interestingly, the non-licensed live vaccine strain, LVS (Type B), lacks both RD18 and RD19 (pilA) due to deletion events mediated by flanking direct repeats. The loss of RD18 and RD19 is responsible for the attenuation of LVS, since re-introducing them in cis could restore the virulence to a level similar to a virulent type B strain. Significantly, these deletion events are irreversible, preventing LVS to revert to a more virulent form. Therefore, this important finding could facilitate the licensing of LVS as a vaccine against tularemia.
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Chen, Chen. "Studies on Selective Protein Loading onto Extracellular Membrane Vesicles of a Novel Cold-Adapted Bacterium, Shewanella vesiculosa HM13." Kyoto University, 2020. http://hdl.handle.net/2433/253331.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第22495号
農博第2399号
新制||農||1076(附属図書館)
学位論文||R2||N5275(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 栗原 達夫, 教授 小川 順, 教授 木岡 紀幸
学位規則第4条第1項該当
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Guschinskaya, Natalia. "Caractérisation moléculaire des signaux de sécrétion des protéines sécrétées par le système de sécrétion de type II de la bactérie phytopathogène Dickeya dadantii." Thesis, Lyon 1, 2014. http://www.theses.fr/2014LYO10085/document.
Full textAbstract:
Le système de sécrétion de type II (T2SS) assure le transport de protéines sous une forme repliée du périplasme dans le milieu extracellulaire. Ce système est largement exploité par les bactéries à Gram négatif pathogènes des plantes, des animaux et de l'homme où il permet la sécrétion de facteurs de virulence (des toxines et des enzymes lytiques). La bactérie phytopathogène Dickeya dadantii utilise le T2SS appelé Out, pour sécréter une douzaine de pectinases qui dégradent les parois des cellules végétales. Les protéines sécrétées par le T2SS n'ont pas de motif de sécrétion apparent et leur sécrétion implique plusieurs interactions transitoires avec les composants du système. La nature moléculaire de ces interactions n'est pas connue. Afin de capter ces interactions transitoires lors du processus de sécrétion, j'ai utilisé le pontage dirigé in vivo. Cette technique repose sur l'incorporation d'un analogue photoréactif d'un acide aminé (le para-benzoyl Lphénylalanine, pBpa) à la place des résidus soupçonnés de faire partie d'un site d'interaction. Le pontage est ensuite activé par une courte exposition des cellules aux UV ce qui permet la formation des complexes protéiques. Tout d'abord, cette technique a été utilisée pour introduire le pBpa dans plusieurs régions exposées à la surface d'une exoprotéine, PelI. Cette stratégie a permis de mettre en évidence qu'un élément structural, la boucle 3 du domaine Fn3 de PelI, est impliquée dans l'interaction avec la sécrétine OutD, le composant du T2SS situé dans la membrane externe, et avec le domaine PDZ d'OutC, un composant de la membrane interne. Ces résultats suggèrent que la boucle 3 fait partie d'un motif de sécrétion. Deux autres régions ont été identifiées au sein de PelI : le linker entre les deux domaines de PelI qui est impliqué dans l'interaction avec OutD et une région exposée du domaine catalytique qui interagit avec la protéine OutC. La même approche a été utilisée pour introduire le pBpa dans les deux composants du T2SS, OutC et OutD. Ces expériences ont suggéré que le domaine PDZ d'OutC interagit avec une autre exoprotéine, PelB. Cette étude, de façon complémentaire à d'autres approches, nous a permis de démontrer certains détails moléculaires essentiels de la sécrétion par le T2SSThe type II secretion system (T2SS) transports folded proteins from the periplasm through the outer membrane into the milieu. In many pathogenic Gram-negative bacteria, the T2SS secretes various virulence factors in host tissue and is directly involved in pathogenesis. The phytopathogen Dickeya dadantii secretes a dozen of pectinases through a T2SS named Out. The secreted proteins are lacking an obvious common signal and secretion is thought to involve multiple transient interactions of folded exoproteins with several T2SS components. Molecular nature of these interactions remains unknown. To address this question we used an in vivo sitespecific photo-crosslinking approach to capture such transient interactions within the functional T2SS of D. dadantii. In this technique, the photo-crosslinker para-benzoyl-L-phenylalanine, pBpa, is introduced in vivo in place of a residue of interest and UV-irradiation of living cells provokes the formation of complexes between the protein of interest and its partners. First, in a systematic approach, pBpa was introduced at several surface-exposed sites of the secreted protein PelI. This strategy permitted us to identify that one structural element, loop 3 of Fn3 domain in PelI, interacts both with the secretin, the outer membrane T2SS component, and with the PDZ domain of OutC, an inner membrane T2SS component. These results suggest that this loop 3 is a part of the secretion motif. The same approach permitted us to identify two other regions of PelI interacting with the T2SS: a linker situated between the two domains of PelI, which interacts with OutD, and an exposed region of the catalytic domain of PelI interacting with OutC. In another approach, pBpa was introduced into the T2SS components, OutC and OutD. These experiments suggested that the PDZ domain of OutC interacts with the secreted protein PelB. This study, in complement with other approaches, allowed us to uncover some important molecular features of the protein secretion by the T2SS
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Cadoret, Frederic. "PA 7, souche atypique de Pseudomonas aeruginosa : Etude transcriptomique et caractérisation d'un troisième système de sécrétion de type II fonctionnel, Txc." Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM4023.
Full textAbstract:
Pseudomonas aeruginosa est une bactérie pathogène opportuniste qui est caractérisée par son ubiquité et sa grande capacité adaptative. Cette faculté lui est notamment permise par de nombreux systèmes de perception et de régulation, la sécrétion d'un large arsenal d'exoprotéines, une capacité à alterner entre deux modes de vie, une haute résistance naturelle aux antibiotiques ainsi qu'un génome riche soumis à une importante plasticité génomique. Cette dernière, associée aux pressions de sélection exercées par la grande diversité d'environnements rencontrés par P. aeruginosa, a permis l'émergence de nombreuses souches aux caractéristiques génotypiques et phénotypiques qui leur sont propres. Durant ma thèse, nous avons réalisé une analyse transcriptomique globale comparative entre les souches connues PA14, PAO1 et un nouvel isolat clinique atypique multirésistant aux antibiotiques, la souche PA7. Cette étude nous a permis de suggérer que cette souche, dépourvue des armes principales de la cytotoxicité, tendait naturellement vers un mode de développement associé à la formation de biofilm. Nous avons également caractérisé l'îlot génomique RGP69, unique à la souche PA7 qui code un troisième système de sécrétion de type II, Txc, qui sécrète dans le milieu extracellulaire une protéine d'affinité à la chitine, CbpE, sous le contrôle régulationnel d'un nouveau système de régulation à deux composants, Tts. Cet îlot génomique serait directement impliqué dans la physiologie particulière de la souche PA7Pseudomonas aeruginosa is an opportunistic bacterial pathogen, characterized by its ubiquity and its high adaptative property. This faculty is particularly due to many systems of perception and regulation, the secretion of a wide arsenal of exoproteins, an ability to switch between two life styles, a high natural resistance to antibiotics and a rich genome submitted to an important genomic plasticity. The latter, combined with the selection pressure exerted by the wide variety of environments encountered by P. aeruginosa, has allowed the emergence of many strains with their own genotypic and phenotypic characteristics.During my thesis, we performed an overall comparative transcriptomic analysis between the known strains PA14 and PAO1, and a new atypical clinical isolate multiresistant to antibiotics, the PA7 strain. This study allowed us to determine that this strain, lacking the main weapons of cytotoxicity, naturally tended to a life-style associated with biofilm formation. We also characterized the RGP69 genomic island, unique in the PA7 strain, which encodes a third type II secretion system, Txc, that secretes in the extracellular medium a chitin-binding protein, CbpE, under the regulatory control of a component system, Tts. This genomic island could be directly involved in the particular physiology of the PA7 strain
Book chapters on the topic "Type II secretion system ; T2SS"
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Korotkov, Konstantin V., and Maria Sandkvist. "Architecture, Function, and Substrates of the Type II Secretion System." In Protein Secretion in Bacteria, 227–44. Washington, DC, USA: ASM Press, 2019. http://dx.doi.org/10.1128/9781683670285.ch19.
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Johnson, Tanya L., Aleksandra E. Sikora, Ryszard A. Zielke, and Maria Sandkvist. "Fluorescence Microscopy and Proteomics to Investigate Subcellular Localization, Assembly, and Function of the Type II Secretion System." In Methods in Molecular Biology, 157–72. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-62703-245-2_10.
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Söderberg, Maria A., and Nicholas P. Cianciotto. "The Type II Protein Secretion System of Legionella pneumophila Is Important for Growth in Iron-Rich Media and Survival in Tap Water at Low Temperatures." In Legionella, 214–16. Washington, DC, USA: ASM Press, 2014. http://dx.doi.org/10.1128/9781555815660.ch53.
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Korotkov, Konstantin V., and Maria Sandkvist. "Architecture, Function, and Substrates of the Type II Secretion System." In Protein Secretion in Bacteria, 227–44. American Society of Microbiology, 2019. http://dx.doi.org/10.1128/ecosalplus.esp-0034-2018.
Full textConference papers on the topic "Type II secretion system ; T2SS"
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Tandon, N. N., and G. A. Jamieson. "ROLE OF PLATELET MEMBRANE GLYCOPROTEIN IV IN PLATELET-COLLAGEN INTERACTION: A MICROTITER ASSAY TO STUDY PLATELET ADHERENCE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643906.
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The role of platelet glycoprotein IV (GPIV) in platelet function has not been elucidated. We have now isolated GPIV (Mr 88,000) from platelet membranes in homogeneous form by a series of steps involving (i) phase partitioning in Triton X-114, (ii) ion exchange chromatography on DEAE-cellulose, (iii) lectin affinity chromatography on WGA-Sepharose, and (iv) size exclusion chromatography on Ultrogel AcA44. Purified GPIV inhibited platelet shape change and aggregation induced by collagen (2 ug/ml; 7 nM as tropocollagen) in a dose-dependent fashion (ID50 ∼ 1 ug/ml; 10 nM) but did not affect aggregation induced by thrombin, ADP, epinephrine, arachidonate or ionophore A23187. To study the role of GPIV in platelet interaction with collagen we have developed a microtiter assay involving (i) coating acid soluble or fibrillar Type I collagen onto microtiter plates, (ii) incubation of coated collagen with 51Cr-labeled platelets and (iii) quantitation of platelet adherence by analysing the radioactivity of the SDS lysate of the adhered platelets. In this assay system, Fab fragments of anti-GPIV antibody inhibited platelet adherence by 75% to both acid soluble and fibrillar Type I collagen while nonimmune serum was without effect. Fab fragments also inhibited collagen-induced aggregation and secretion (ID∼ 10 ug/ml; 200 nM) and, slightly less effectively, aggregation by ADP and epinephrine (ID∼ 300 NM), but did not affect platelet activation by thrombin, arachidonate or ionophore. Fab fragments also inhibited platelet attachment to collagen-Sepharose columns by 80%. These results suggest a role for GPIV in the interaction of platelets with collagen, probably at the level of primary platelet adherence.