The conformation of nascent polylysine and polyphenylalanine peptides on ribosomes

Polypeptide synthesis using either phenylalanine or lysine was initiated on Escherichia coli ribosomes; then the position and conformation of the nascent peptide were monitored by fluorescence techniques. To this end, fluorophores had been attached to the amino terminus of each nascent peptide, and major differences were observed as chain extension occurred. Polyphenylalanine appeared to build up as a hydrophobic mass adjacent to the peptidyl transferase center while polylysine apparently was extended directly from the ribosome into the surrounding solution. An explanation for these differences may be provided by the physical and chemical properties of each polypeptide. These properties may be responsible for the route by which each peptide exits the peptidyl transferase center as demonstrated by the different sensitivity of each to inhibition by erythromycin

Liposomes Recruit IpaC to the Shigella flexneri Type III Secretion Apparatus Needle as a Final Step in Secretion Induction

Shigella flexneri contact with enterocytes induces a burst of protein secretion via its type III secretion apparatus (TTSA) as an initial step in cellular invasion. We have previously reported that IpaD is positioned at the TTSA needle tip (M. Espina et al., Infect. Immuno. 74:4391-4400, 2006). From this position, IpaD senses small molecules in the environment to control the presentation of IpaB to the needle tip. This step occurs without type III secretion induction or IpaC recruitment to the S. flexneri surface. IpaC is then transported to the S. flexneri surface when target cell lipids are added, and this event presumably mimics host cell contact. Unlike IpaB mobilization, IpaC surface presentation is closely linked to secretion induction. This study demonstrates that sphingomyelin and cholesterol are key players in type III secretion induction and that they appear to interact with IpaB to elicit IpaC presentation at the TTSA needle tip. Furthermore, IpaB localization at the needle tip prior to membrane contact provides the optimal set of conditions for host cell invasion. Thus, the S. flexneri type III secretion system can be induced in a stepwise manner, with the first step being the stable association of IpaD with the needle tip, the second step being the sensing of small molecules by IpaD to mobilize IpaB to the tip, and the third step being the interaction of lipids with IpaB to induce IpaC localization at the needle tip concomitant with translocon insertion into the host membrane and type III secretion induction. Shigella flexneri, the causative agent of shigellosis, is responsible for more than 1 million deaths each year, especially among children in developing regions (www.who.int/vaccines-documents/DocsPDF99/www9947.pdf). Once ingested, S. flexneri crosses M cells and passes into the underlying gut-associated lymphoid tissues of the colon (20), where it kills macrophages (29) and then invades epithelial cells by macropinocytosis (17). The S. flexneri invasive phenotype localizes genetically to a 31-kb region of its large virulence plasmid and is absolutely tied to its type III secretion system (TTSS) (6, 23). TTSSs are used by numerous gram-negative bacteria to introduce bacterially derived effector proteins into the membrane and cytoplasm of a target cell, resulting in the subversion of normal cell functions (8). Linking the bacterium and host cell in this process is the type III secretion apparatus (TTSA), which structurally resembles a molecular needle and syringe. The system is controlled by a basal body (the syringe) that spans both bacterial membranes and an external needle that provides a conduit from the basal body to the sensory needle tip complex (8, 28). The needle in S. flexneri is comprised of a polymer of MxiH and is approximately 50 nm long and 7 nm in diameter, with a central channel that is about 2.5 nm in diameter (5). At the top of the MxiH needle resides the tip protein IpaD, most likely as a pentamer, which serves as an environmental sensor for the MxiH-IpaD tip complex (4, 7). When the presence of bile salts such as deoxycholate (DOC) is sensed by IpaD, the first translocator protein, IpaB, is mobilized to the TTSA needle tip to form an MxiH-IpaD-IpaB ternary complex. At this stage, the TTSA structure is primed for subsequent host cell contact (19, 24). In previous studies IpaC had not been found to localize to the S. flexneri surface of the log-phase bacterium (7, 19). As a next step in describing the process of type III secretion, we show here that liposomes trigger mobilization of IpaC to the needle tip complex, where it is immediately inserted into the host cell membrane, along with IpaB, to complete the TTSA conduit into the host cell just prior to initiating host cytoskeleton rearrangements. IpaC is most efficiently recruited to the S. flexneri surface with a defined liposome composition that includes phospholipids, sphingomyelin (SM), and cholesterol (Chol). Furthermore, IpaC recruitment occurs concomitantly with induction of type III secretion of IpaB, IpaC, and IpaD into the S. flexneri culture supernatant. It thus appears that IpaB mobilization to the S. flexneri TTSA needle tip represents a second discrete step in TTSA assembly, with the final third step being IpaC recruitment to the needle tip, which occurs after IpaB contacts and inserts into the host cell membrane

Impact of Detergent on the Biophysical Properties and Immune Response of the IpaDB Fusion Protein, a Candidate Subunit Vaccine against Shigella spp.

Shigella spp. are causative agents of bacillary dysentery, a human illness with high global morbidity levels, particularly among elderly and infant populations. Shigella infects via the fecal-oral route, and its virulence is dependent upon a type III secretion system (T3SS). Two components of the exposed needle tip complex of the Shigella T3SS, invasion plasmid antigen D (IpaD) and IpaB, have been identified as broadly protective antigens in the mouse lethal pneumonia model. A recombinant fusion protein (DB fusion) was created by joining the coding sequences of IpaD and IpaB. The DB fusion is coexpressed with IpaB\u27s cognate chaperone, IpgC, for proper recombinant expression. The chaperone can then be removed by using the mild detergents octyl oligooxyethelene (OPOE) or N,N-dimethyldodecylamine N-oxide (LDAO). The DB fusion in OPOE or LDAO was used for biophysical characterization and subsequent construction of an empirical phase diagram (EPD). The EPD showed that the DB fusion in OPOE is most stable at neutral pH below 55°C. In contrast, the DB fusion in LDAO exhibited remarkable thermal plasticity, since this detergent prevents the loss of secondary and tertiary structures after thermal unfolding at 90°C, as well as preventing thermally induced aggregation. Moreover, the DB fusion in LDAO induced higher interleukin-17 secretion and provided a higher protective efficacy in a mouse challenge model than did the DB fusion in OPOE. These data indicate that LDAO might introduce plasticity to the protein, promoting thermal resilience and enhanced protective efficacy, which may be important in its use as a subunit vaccine

IpaD Localizes to the Tip of the Type III Secretion System Needle of Shigella flexneri

This is the publisher's version, also available electronically from http://iai.asm.org/content/74/8/4391Shigella flexneri, the causative agent of shigellosis, is a gram-negative bacterial pathogen that initiates infection by invading cells within the colonic epithelium. Contact with host cell surfaces induces a rapid burst of protein secretion via the Shigella type III secretion system (TTSS). The first proteins secreted are IpaD, IpaB, and IpaC, with IpaB and IpaC being inserted into the host cell membrane to form a pore for translocating late effectors into the target cell cytoplasm. The resulting pathogen-host cross talk results in localized actin polymerization, membrane ruffling, and, ultimately, pathogen entry. IpaD is essential for host cell invasion, but its role in this process is just now coming to light. IpaD is a multifunctional protein that controls the secretion and presentation of IpaB and IpaC at the pathogen-host interface. We show here that antibodies recognizing the surface-exposed N terminus of IpaD neutralize Shigella's ability to promote pore formation in erythrocyte membranes. We further show that MxiH and IpaD colocalize on the bacterial surface. When TTSS needles were sheared from the Shigella surface, IpaD was found at only the needle tips. Consistent with this, IpaD localized to the exposed tips of needles that were still attached to the bacterium. Molecular analyses then showed that the IpaD C terminus is required for this surface localization and function. Furthermore, mutations that prevent IpaD surface localization also eliminate all IpaD-related functions. Thus, this study demonstrates that IpaD localizes to the TTSA needle tip, where it functions to control the secretion and proper insertion of translocators into host cell membrane

IpaD Localizes to the Tip of the Type III Secretion System Needle of Shigella flexneri

This is the publisher's version, also available electronically from http://iai.asm.org/content/74/8/4391Shigella flexneri, the causative agent of shigellosis, is a gram-negative bacterial pathogen that initiates infection by invading cells within the colonic epithelium. Contact with host cell surfaces induces a rapid burst of protein secretion via the Shigella type III secretion system (TTSS). The first proteins secreted are IpaD, IpaB, and IpaC, with IpaB and IpaC being inserted into the host cell membrane to form a pore for translocating late effectors into the target cell cytoplasm. The resulting pathogen-host cross talk results in localized actin polymerization, membrane ruffling, and, ultimately, pathogen entry. IpaD is essential for host cell invasion, but its role in this process is just now coming to light. IpaD is a multifunctional protein that controls the secretion and presentation of IpaB and IpaC at the pathogen-host interface. We show here that antibodies recognizing the surface-exposed N terminus of IpaD neutralize Shigella's ability to promote pore formation in erythrocyte membranes. We further show that MxiH and IpaD colocalize on the bacterial surface. When TTSS needles were sheared from the Shigella surface, IpaD was found at only the needle tips. Consistent with this, IpaD localized to the exposed tips of needles that were still attached to the bacterium. Molecular analyses then showed that the IpaD C terminus is required for this surface localization and function. Furthermore, mutations that prevent IpaD surface localization also eliminate all IpaD-related functions. Thus, this study demonstrates that IpaD localizes to the TTSA needle tip, where it functions to control the secretion and proper insertion of translocators into host cell membrane

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

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

Virologie / Bakteriologie / Mykologie

141 - Effizienz von Kaliumhypochlorit zur Inaktivierung ausgewählter pilzlicher, bakterieller und viraler PflanzenkrankheitserregerEfficancy of Potassium Hypochlorite (KClO) to inactivate selected plant pathogenic fungi, bacteria and virusesMarlon-Hans Rodríguez, Martina Bandte, Gerhard Fischer, Carmen Büttner142 - Eignung von elektrolytisch generiertem Kaliumhypochlorit zur Inaktivierung von Pflanzenviren in rezirkulierender Nährlösungen im Gewächshausanbau von TomatenAbility of electrolysed produced Potassium Hypochlorite (KClO) to inactivate plant viruses in recirculating nutrient solutions in greenhouse production of tomatosJanine Paulke, Martina Bandte, Carmen Büttner143 - Ultrafiltration und Ultrazentrifugation zur Konzentrierung von Pflanzenviren in NährlösungUltrafiltration and ultracentrifugation as tools to concentrate plant viruses in nutrient solutionJanina Vincenz, Martina Bandte, Carmen Büttner144 - Reinigung doppelsträngiger RNA in Verbindung mit Hochdurchsatzsequenzierung als Werkzeug zum Nachweis von RNA Viren in PflanzenThe combination of double-stranded RNA isolation and deep sequencing as an unspecific diagnostic tool to assess the presence of RNA viruses in plantsTill Lesker, Paul Rentz, Edgar Maiss145 - Impact of silica supplementation on virus infected cucumber culturesRolle der Kieselsäureapplikation Virus infizierter GurkenkulturenSabine Holz, Grzegorz Bartoszewski , Michael Kube, Carmen Büttner146 - Untersuchungen zum Auftreten des Arabis mosaic virus in Birken aus Rovaniemi (Finnland) mit Virus-spezifischen SymptomenInvestigations on the occurence of Arabis mosaic virus in birches from Rovaniemi (Finland) with virus-specific symptomsRichard Pauwels, Markus Rott, Susanne von Bargen, Carmen Büttner147 - Cherry leaf roll virus in Betula spp. in Finland: what do we know about its population diversity?Cherry leaf roll virus in Birken-Arten in Finnland: Was wissen wir über die Populationsdiversität?A. Rumbou, S. von Bargen, M. Rott, R. Jalkanen, C. Büttner148 - Viruserkrankungen im WeinbauViroses in viticultureHenriette Gruber, Patricia Bohnert, Christiane Rieger149 - Molecular analysis of Tobacco rattle virus isolates from potatoes in various parts of GermanyKerstin Lindner, Renate Koenig150 - Detektion und Diversität des European mountain ash ringspot-associated virus (EMARaV) in Ebereschen (Sorbus aucuparia L.) in NorwegenDetection and variability of European mountain ash ringspot-associated virus (EMARaV) in Sorbus aucuparia L. in NorwayTheresa Büttner, Jenny Robel, Hans-Peter Mühlbach, Susanne von Bargen, Carmen Büttner151 - Charakterisierung des European mountain ash ringspot-associated virus (EMARaV) in Mehlbeerenarten (Sorbus spp.)Characterization of the European mountain ash ringspot-associated virus (EMARaV) in whitebeam species (Sorbus spp.)Luisa Dieckmann, Jenny Robel, Susanne von Bargen, Carmen Büttner152 - Vollständige Genomsequenz eines Carrot virus S Isolates aus Meerfenchel aus SpanienW. Menzel, P. Menzel, S. Winter153 - Nachweis und vollständige Sequenzierung eines Carla- und eines Potex-virus aus Epiphyullum spec.Detection and complete sequence of a Carla- and Potexvirus in Epiphyllum spec.Edgar Maiss, Paul Rentz, Annette Hohe, Rosa Herbst154 - Analysis of mixed populations of latent viruses of apple and rubbery wood disease of apple using new generation sequencingAnalyse von Mischpopulationen latenter Apfelviren und der Gummiholzkrankheit an Apfel mittels HochdurchsatzsequenzierungVladimir Jakovljevic, Patricia Otten, Jonathon Blake, Wilhelm Jelkmann155 - Experiments on transmission of viroids under glass and longevity of viroid RNA in detached leaves under different storage conditionsThi Thu Vo, Heinz-Wilhelm Dehne, Stephan Winter, Joachim Hamacher156 - Phytoplasmen in Schleswig-HolsteinPhytoplasmas in the state of Schleswig-HolsteinG. Henkel, C. Willmer, M. Wunderlich, B. Golecki157 . Phytoplasmen verändern das Dufststoffbouquet ihres pflanzlichen LebensraumsPlant volatile emission is affected by phytoplasma infectionMargit Rid, Kai Lukat, Svenja Hoferer, Jürgen Gross159 - Ist das Wurzelbild ein Sortierungsmerkmal für durch Candidatus Phytoplasma pyri verursachten Birnenverfall?Is the root file a sorting feature for Pear decline caused by Canditatus Phytoplama pyri?Georg Henkel, Claudia Willmer, Bernd Kaland, Bettina Golecki160 - Die Bedeutung von β-Caryophyllen als Lockstoff für die Apfeltriebsucht übertragende Blattsaugerart Cacopsylla pictaThe impact of β-caryophyllene as attractant for the Apple Proliferation transmitting insect Cacopsylla pictaConstanze Mesca, Svenja Hoferer, Jürgen Gross161 - Echte Mehltauarten an Beet- und BalkonpflanzenSpecies of powdery mildews on bedding plantsUlrike Brielmaier-Liebetanz162 - Echter Mehltau an Petersilie – Untersuchungen zum WirtspflanzenspektrumPowdery Mildew of Parsley – studies on the host rangePeggy Marx, Ute Gärber163 - Falscher Mehltau an Petersilie – Untersuchungen zum Wirtspflanzenspektrum und molekularbiologische CharakterisierungDowny mildew of parsley – studies on the host range and molecular characterizationGabriele Leinhos, Hermann-Josef Krauthausen, Frank Brändle164 - Welkekrankheit an Euonymus japonicaWilt disease on Euonymus japonicaUlrike Brielmaier-Liebetanz, Roswitha Ulrich, Stefan Wagner, Sabine Werres165 - Taxonomische Analyse der mikrobiellen Gemeinschaft von Zuckerrüben unter unterschiedlichen Lagerbedingungen mittels Hochdurchsatz-Amplikonsequenzierung von unterschiedlichen MarkergenenTaxonomic analysis of the microbial community in stored sugar beets using highthroughput sequencing of different marker genesSebastian Liebe, Daniel Wibberg, Anika Winkler, Alfred Pühler, Andreas Schlüter, Mark Varrelmann166 - Molecular characterization of a novel mycovirus found in Rhizoctonia solani AG 2-2IIIBMolekulare Charakterisierung eines neuen Mycovirus aus Rhizoctonia solani AG 2-2 IIIBAnika Bartholomäus, Mark Varrelman

Antibody response of monkeys to invasion plasmid antigen D after infection with Shigella spp.

The antigen preparation most often used for determining the levels of antibodies to virulence-associated proteins of Shigella spp. consists of a mixture of proteins (including IpaB, IpaC, IpaD, and VirG*) extracted from virulent shigellae with water (water extract). To overcome the lack of specificity for individual antigens in the water-extract enzyme-linked immunosorbent assay (ELISA), the ipaD gene from S. flexneri has been cloned, expressed to a high level, and purified for use in a new ELISA for the determination of the levels of antibody against IpaD in monkeys and humans challenged with shigellae. The IpaD ELISA for serum immunoglobulins G and A correlated well with the water-extract ELISA in that monkeys infected with S. flexneri or S. sonnei responded with high serum antibody titers in both assays. The IpaD assay required less antigen per well, had much lower background levels, and did not require correction with antigens from an avirulent organism. In conjunction with the water-extract ELISA, it was possible to identify infected animals that did not respond to IpaD but did produce antibodies that reacted in the water-extract ELISA. This indicates that even though IpaB, IpaC, and IpaD are essential for the invasiveness phenotype, the infected host does not always produce antibodies against all components of the invasiveness apparatus

Soluble invasion plasmid antigen C (IpaC) from Shigella flexneri elicits epithelial cell responses related to pathogen invasion.

Shigella flexneri invades colonic epithelial cells by pathogen-induced phagocytosis. The three proposed effectors of S. flexneri internalization are invasion plasmid antigens B (IpaB), IpaC, and IpaD, which are encoded on the pathogen's 230-kb virulence plasmid and translocated to the extracellular milieu via the Mxi-Spa translocon. To date, there are no definitive functional data for any purified Ipa protein. Here, we describe the first characterization of highly purified recombinant IpaC, which elicits numerous epithelial cell responses related to events that take place during pathogen invasion. 125I-labeled IpaC binds cultured Henle 407 intestinal cells with an apparent dissociation constant in the low micromolar range. Moreover, incubation of epithelial cells with IpaC results in general changes in cellular phosphoprotein content, demonstrating this protein's ability to influence cellular protein kinase activities. These results contrast dramatically with those seen for recombinant IpaD, which does not bind to or induce detectable changes in the normal activities of cultured epithelial cells. In addition to influencing host cell activities, preincubation of epithelial cells with purified IpaC enhances uptake of S. flexneri by host cells. A similar result is seen when the cells are preincubated with a highly concentrated water extract of virulent S. flexneri 2a (strain 2457T). Interestingly, soluble IpaC also appears to promote uptake of the noninvasive S. flexneri 2a strain BS103. Purified IpaD failed to enhance the uptake of virulent S. flexneri and did not facilitate uptake of BS103. Taken together, the data suggest that IpaC is a potential effector of the host cell biological activities and may be responsible for entry of S. flexneri into target cells

A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by fluorescence measurements: comparison to a synthetic alanyl-elongator tRNA.

Two synthetic tRNAs have been generated that can be enzymatically aminoacylated with alanine and have AAA anticodons to recognize a poly(U) template. One of the tRNAs (tRNA(eAla/AAA)) is nearly identical to Escherichia coli elongator tRNA(Ala). The other has a sequence similar to Escherichia coli initiator tRNA(Met) (tRNA(iAla/AAA)). Although both tRNAs can be used in poly(U)-directed nonenzymatic initiation at 15 mM Mg2+, only the elongator tRNA can serve for peptide elongation and polyalanine synthesis. Only the initiator tRNA can be bound to 30S ribosomal subunits or 70S ribosomes in the presence of initiation factor 2 (IF-2) and low Mg2+ suggesting that it can function in enzymatic peptide initiation. A derivative of coumarin was covalently attached to the alpha amino group of alanine of these two Ala-tRNA species. The fluorescence spectra, quantum yield and anisotropy for the two Ala-tRNA derivatives are different when they are bound to 70S ribosomes (nonenzymatically in the presence of 15 mM Mg2+) indicating that the local environment of the probe is different. Also, the effect of erythromycin on their fluorescence is quite different, suggesting that the probes and presumably the alanine moiety to which they are covalently linked are in different positions on the ribosomes